Method for Controlling Hematophagous or Sap-Feeding Arthropods

ABSTRACT

Modulation of inward potassium ion conductance with structurally diverse small-molecules in the arthropod salivary gland induces arthropod salivary gland failure that results in a reduction or elimination in the ability of the arthropod to feed. Administering Kir channel inhibitors reduces food intake, increases feeding time, reduces salivary gland secretion, induces mortality, and reduces transmission of vector-borne pathogens. Kir channel inhibitors induce these adverse effects in ticks, mosquitoes, horn flies, and aphids.

The benefit of the 14 Nov. 2016 filing date of U.S. provisional patentapplication Ser. No. 62/421,621 is claimed; and the benefit of the 15Nov. 2016 filing date of U.S. provisional patent application Ser. No.62/422,382 is claimed; this benefit is claimed under 35 U.S.C. § 119(e)in the United States, and under applicable treaties and conventions inall countries.

This invention was made with United States Government support undergrant number 58-3094-5-016 awarded by the Department of Agriculture,Agricultural Research Service; a grant from the Foundation for theNational Institutes of Health through the Vector-Based Transmission ofControl: Discovery Research (VCTR) program of the Grand Challenges inGlobal Health initiative; and grant no. 1R01DK082884 from the NationalInstitute of Diabetes and Digestive and Kidney Diseases. The UnitedStates Government has certain rights in this invention.

TECHNICAL FIELD

This invention pertains to a method for controlling hematophagous orsap-feeding arthropods.

BACKGROUND ART

Mosquitoes are vectors of devastating human pathogens such as Zikavirus, malaria parasites, and dengue fever virus. Insecticides have beenthe primary tools for controlling the spread of mosquito-borne diseases,but the emergence of insecticide-resistant populations of mosquitoes isthreatening the effectiveness of these control agents across the world.Mosquitoes are vectors for pathogens that impose enormous health andsocioeconomic burdens, particularly in the developing world. The malariavector An. gambiae and the dengue/yellow fever vector Ae. aegypti arecollectively responsible for hundreds of millions of cases of malariaand dengue fever annually, leading to over 500,000 deaths per year.Moreover, Ae. aegypti is suspected as the primary vector in the recentoutbreak of Zika virus in Latin America and the Caribbean; Zika virushas been linked to dramatic increases in the number of cases ofmicrocephaly and Guillan-Barré syndrome. The two major classes ofinsecticides that have been used in vector control programs arepyrethroids and anticholinergics (carbamates/organophosphates). Theseagents work by, respectively, blocking inactivation of voltage-gatedsodium channels or inhibiting acetylcholinesterase enzymes expressed inthe nervous system. Moreover, they act on all developmental stages andsexes, creating intense selective pressure for target site resistance(e.g., knockdown resistance, kdr) and/or metabolic resistance (e.g.,elevated expression of cytochrome P450 monoxygenases). The developmentof resistance and the lack of novel, validated target sites that can beexploited for mosquito control complicate efforts to mitigate the spreadof emerging mosquito-borne pathogens around the globe, including Zikaand chikungunya viruses. There is an unfilled need for new classes ofinsecticides, acting on new molecular targets, to bolster integratedvector control programs and limit the spread of mosquito-borne diseases.

Tick-borne pathogens are ubiquitously present throughout the world,presenting significant concerns to global health and agriculturalproductivity. Ticks are second only to mosquitoes as disease vectors.Ticks transmit a variety of infectious bacteria, viruses, protozoa,fungi, and helminthes. In addition to transmitting pathogens, somespecies of ticks produce a toxin that causes paralysis, termed “tickparalysis.” The host immune response to tick attachment can cause skindamage and pain. From a veterinary perspective, ticks are responsiblefor significant economic losses stemming from tick-caused anemia,reducing growth rate, reducing milk production, and increasing mortalityfrom pathogen infection. For humans, the number of cases of Lymeborreliosis reported to the Centers for Disease Control and Prevention(CDC) has nearly tripled over the past 20 years, with more than 28,000confirmed cases for the year 2015, despite extensive research effortsaimed at controlling this disease. Rickettsial disease cases aresteadily increasing in the United States. Some strains of Rickettsiarickettsii, the causative agent of Rocky Mountain Spotted Fever, cancause significant mortality in humans. The primary arthropod vectors ofR. rickettsii are Dermacentor andersoni and D. variabilis, which rarelyfeed on humans. However, recent studies have shown that the mostpredominant human biting tick, Amblyomma americanum, is capable ofacquiring, maintaining, and transmitting R. rickettsii as well. Tickcontrol has been primarily based on neurotoxic acaricides, but extensiveuse has resulted in resistant tick populations. The continuing increasein tick populations and their associated pathogens underlines thefailure of current control measures.

Classical synthetic insecticides to control flies, ticks, mosquitoes,aphids, and other unwanted insects or arachnids have targeted suchthings as sodium channels or acetylcholinesterases. Classicalinsecticides suffer from disadvantages such as the development of highlevels of insecticide resistance in target species, and effects onnon-target species such as honey bees.

The molecular architecture of arthropod salivary glands has beenexamined in some organisms including fruit flies, mosquitoes, ticks,fleas, and black flies. It is known that saliva constituents arerequired for blood feeding through regulating vasodilation, regulatingblood clotting, acting as anesthetics, and providing anti-immunefactors. Despite this work, the understanding of the molecular machineryand physiological systems of arthropod salivary glands remains limited.

Tick salivary glands have perhaps been the most commonly studied.Dopamine receptors have been reported in the salivary glands of theblacklegged tick (Ixodes scapularis), receptors that control inwardfluid transport and release of fluid to coordinate salivary secretion.Pharmacological evidence suggests the dopaminergic system is a majorphysiological pathway in arthropod salivation. However, informationregarding the ion transport pathways of arthropod salivary glands,including ticks, is extremely limited.

K⁺ ion transport in mammalian salivary glands is critical for generatingsaliva. Inwardly rectifying potassium (Kir) channels have been shown tobe essential to mammalian salivary gland function. These channelsfunction as “biological diodes” by favoring the flow of potassium ionsinwardly rather than outwardly. Known Kir channels share a similarmolecular structure: They are tetramers assembled around amembrane-spanning pore, and are gated by polyvalent cations that occludethe pore at cell potentials more positive than the K⁺ equilibriumpotential (E_(k)). The number of genes encoding Kir channel componentsvaries by species, with humans having 16 Kir channel-encoding genes,Aedes aegypti mosquito having 5, and D. melanogaster having 3. TheDrosophila Kir genes are termed Ir, Irk2, and Irk3, and encode Kir1,Kir2, and Kir3, respectively. Tissue expression patterns of DrosophilaKir channels are highly variable. There are three sub-families of Kirchannels: classical, ATP-sensitive (K_(ATP)), and GPCR-gated. ClassicalKir channels are constitutively active, and depend on membrane potentialto induce inward rectification. ATP-sensitive Kir channels are gated bythe presence or absence of nucleotides (ATP, ADP), and are closed in thepresence of ATP. These channels are heteromeric, comprising 4 Kirchannel subunits and 4 sulphonylurea receptors that are responsible forbinding the ATP or small molecule activators/inhibitors. GPCR-gated Kirchannels are diverse, and little is known about their function orbiophysics in arthropods.

Kir channels play important physiological roles in the exocrine systemsof dipteran insects. In D. melanogaster, embryonic depletion of Kir1 andKir2 mRNA in Malpighian tubules significantly reduces transepithelialsecretion of fluid and K⁺ transport. In Aedes aegypti (Ae or Ae.aegypti) and Anopheles gambiae (An or An. gambiae) mosquitoes,pharmacological inhibition of AeKir1 or AnKir1 with structurallydistinct small molecules (viz., VU573, VU590, VU041) disrupts thesecretion of fluid and K⁺ in isolated Malpighian tubules, it impairsdiuretic capacity in adult females, and it impairs K⁺ homeostasis inadult females. See R. Raphemot, M. F. Rouhier, C. R. Hopkins, R. D.Gogliotti, K. M. Lovell, R. M. Hine, D. Ghosalkar, A. Longo, K. W.Beyenbach, J. S. Denton, P. M. Piermarini, Eliciting renal failure inmosquitoes with a small-molecule inhibitor of inward-rectifyingpotassium channels. PloS one 8, e64905(2013)10.1371/journal.pone.0064905, R. Raphemot, M. F. Rouhier, D. R.Swale, E. Days, C. D. Weaver, K. M. Lovell, L. C. Konkel, D. W. Engers,S. F. Bollinger, C. Hopkins, P. M. Piermarini, J. S. Denton, Discoveryand characterization of a potent and selective inhibitor of Aedesaegypti inward rectifier potassium channels. PloS one 9, e110772(2014)10.1371/journal.pone.0110772); M. F. Rouhier, P. M. Piermarini,Identification of life-stage and tissue-specific splice variants of aninward rectifying potassium (Kir) channel in the yellow fever mosquitoAedes aegypti. Insect biochemistry and molecular biology 48, 91-99(2014); published online Epub May (10.1016/j.ibmb.2014.03.003); D. R.Swale et al., An insecticide resistance-breaking mosquitocide targetinginward rectifier potassium channels in vectors of Zika virus andMalaria. Scientific Reports vol. 6, article no. 36954 (published online16 Nov. 2016).

In Drosophila Kin expression is increased by 37-fold in the salivaryglands of both larvae and adults. See Z. Luan, H. S. Li, Inwardlyrectifying potassium channels in Drosophila. Sheng li xue bao: [Actaphysiologica Sinica] 64, 515-519 (2012); published online EpubOct 25; V.R. Chintapalli, J. Wang, J. A. Dow, Using FlyAtlas to identify betterDrosophila melanogaster models of human disease. Nature genetics 39,715-720 (2007); published online EpubJun (10.1038/ng2049).

D. Kim et al., “Multiple functions of Na/K-ATPase in dopamine-inducedsalivation of the blacklegged tick, Ixodes scapularis,” ScientificReports, vol. 6, report no. 21047 (2016) disclose data suggesting thatNa/K-ATPase is involved in dopamine-mediated salivary secretion inticks.

See also: R. Raphemot et al., “Discovery and characterization of apotent and selective inhibitor of Aedes aegypti inward rectifierpotassium channels,” PLOS ONE 9(11), e110772 (2014); R. Raphemot et al.,“Discovery, characterization, and structure-activity relationships of aninhibitor of inward rectifier potassium (Kir) channels with preferencefor Kir2.3, Kir3.X, and Kir7.1,” Frontiers in Pharmacology, vol. 2,article 75 (2011); and L. Lewis et al., “High-throughput screeningreveals a small-molecule inhibitor of the renal outer medullarypotassium channel and Kir7.1,” Molecular Pharmacology,” vol. 76, pp.1094-1103 (2009).

D. Swale et al., “Role of inward rectifier potassium channels insalivary gland function and sugar feeding of the fruit fly, Drosophilamelanogaster,” Pesticide Biochemistry and Physiology, vol. 141, pp.41-49 (2017; epub 15 Nov. 2016) reports on related work, published bythe present inventors and their colleagues.

DISCLOSURE OF THE INVENTION

The arthropod salivary gland is critical in feeding processes, but thesalivary gland has not received much attention in prior methods ofcontrolling arthropod pests. The salivary gland provides constituentsneeded for blood feeding and sap feeding (e.g. anticoagulants, otherenzymes). The salivary gland is the site of osmoregulation, and it isthe site of pathogen transmission.

We have discovered the critical role potassium ion transport pathwaysserve in arthropod feeding and in ultimate pathogen transmission. Wehave discovered a novel target site to inhibit arthropod feeding, toinhibit arthropod salivation, or both. We have confirmed these effectswith several inhibitors of the target site. Novel small-moleculemodulators inhibit or activate Kir channels in salivary glands,inhibiting or ending the arthropod's ability to salivate or feed.Genetic knockdown experiments have helped rule out the possibility thatthe observations might have resulted from off-target effects of thecompounds.

In arthropod salivary glands and gustatory organs, potassium ionconductance pathways transmit potassium ions both inwardly (e.g. inwardrectifier potassium (Kir) channels), and outwardly (two-pore domainpotassium (K2P) channels; calcium activated potassium channel). We haveused small molecules, such as VU041 and pinacidil, to demonstrate thephysiological role of these channels in arthropod salivary glandfunction and feeding. Our observations showed that these potassium ionconductance pathways are essential for proper salivary gland functionand gustatory processes during feeding in Drosophila flies, two speciesof ticks, Aedes aegypti mosquitoes, Haematobia irritans irritans (hornfly), and the cotton aphid, Aphis gossypii. We expect these findings toextrapolate to other species of blood-feeding and sap-feeding insectsand arachnids as well, including other species of mosquitoes, flies,aphids, and ticks.

We have discovered that modulation of inward K⁺ ion conductance in thearthropod salivary gland will induce arthropod salivary gland failure,and will reduce arthropod feeding capabilities; and we have identifiedcompounds that will accomplish this goal. Our data show thatadministering Kir channel inhibitors (or genetic depletion of Kirchannels specifically in the salivary gland) reduces food intake andincreases feeding time in Drosophila melanogaster. Our data show thatpharmacological modulation of Kir channels (K_(ATP)) eliminates salivarygland secretion and reduces or prevents blood ingestion by the tickAmblyomma americanum. Our data show that topical exposure or ingestionof Kir channel modulators while blood feeding prevents blood ingestionby Aedes aegypti mosquitoes in 97.9% of mosquitoes. and reduces intakeby 99.9% in the remaining 2.1%. Our data show that Kir modulation withdiverse scaffolds eliminates the ability of horn flies to ingest blood.Our data show that topical exposure of Kir channel modulators by treatedcotton leaves eliminates all feeding phases (ingestion, egestion,salivation) in the cotton aphid. Our data show that modulation of inwardK⁺ ion conductance in the arthropod salivary gland will induce mortalitythrough an inability to osmoregulate. Our data show that that modulationof inward K⁺ ion conductance in the arthropod salivary gland reducestransmission of vector-borne pathogens

We have discovered that VU041 and other classical Kir channel modulatorsblock feeding and prevent salivation in non-blood feeding arthropods. Wehave discovered that pinacidil, VU063, and other modulators ofATP-sensitive Kir channels block feeding and prevent salivation in bloodfeeding arthropods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A) and 1(B) depict the structures of VU041 and VU937,respectively.

FIG. 2(A) depicts cumulative consumption of sucrose solution byDrosophila 1, 2, 3, and 4 days post-emergence. Controls are designatedby solid circles, dopamine treatment by open squares, and fluphenazinetreatment by open circles. FIG. 2(B) depicts cumulative consumption ofsucrose solution by Drosophila 1, 2, 3, and 4 days post-emergence.Controls are designated by solid circles, VU041 treatment by opensquares, and VU937 treatment by open circles. FIG. 2(C) depicts aconcentration-response curve comparing total consumption valuescollected on day 4 for VU041 (closed circles) and VU937 (open square).FIG. 2(D) depicts the effects of added extracellular potassium toVU041-mediated reduction of consumption. Control (upper solid line) andVU041 (200 μM; lower solid line) traces are duplicated from FIG. 2(A)for comparison to consumption with 300 μM potassium alone (trace withopen circles) and 500 μM potassium+300 μM VU041 (trace with open square)traces.

FIG. 3 displays the average fold-change in Drosophila mRNA levelsrelative to that in wild type Oregon-R control salivary glands. The leftbar is wild type; the middle bar is GFP-KD (control); and the right baris irk1-KD.

FIG. 4(A) depicts cumulative consumption of sucrose solution byDrosophila 1, 2, 3, and 4 days post-emergence. Controls are the middle,solid circles. GFP knockdown are the open squares, and irk1 knockdownare the lower, solid circles. FIG. 4(B) depicts cumulative feeding timefor sucrose solution by Drosophila 1, 2, and 3 days post-emergence.Controls are the left bar on each day; GFP knockdown are the middle baron each day, and irk1 knockdown are the right bar on each day. Asterisksindicate results whose difference from control is statisticallysignificant (P<0.05).

FIG. 5 depicts the structures of compounds VU041, VU937, and VU 730.

FIGS. 6(A) and 6(B) depict the dose-mortality relationship for VU041against different strains of mosquitoes, as indicated in the respectivelegends. FIG. 6(C) depicts abdominal diameters of mosquitoes treatedwith vehicle, control, or VU041 as a function of time elapsed followingblood feeding. FIG. 6(D) depicts diuresis of mosquitoes treated withvehicle, control, or VU041.

FIGS. 7(A) and 7(B) depict the number of eggs laid per female for themosquito species Anopheles gambiae and Aedes aegypti, respectively,following treatment with vehicle, control, and VU041.

FIGS. 8(A) and 8(B) depict the mortality of control versus VU041, and ofcontrol versus bifenthrin, respectively, on adult honey bees.

FIG. 9 depicts a synthesis of the compound VU041.

FIGS. 10(A)-(D) depict the influence of Kir channel blockage on fluid-and ion-secretion in the isolated tick salivary gland. FIG. 10(A)depicts a fluid secretion assay using a whole salivary gland withdopamine (DA) and the Kir channel blocker barium chloride. FIGS. 10(B),(C), and (D) depict ion secretion rates for sodium, chloride, andpotassium, respectively, following dopamine exposure (control) or Kirchannel blockage (BaCl₂).

FIGS. 11(A)-(C) depict fluid secretion rates in isolated tick salivaryglands after pharmacological modulations (as indicated) of K_(ATP)channels, K2P channels, and the KCC transporter, respectively.

FIG. 12 depicts the percentage of mosquitoes able to take a blood mealin an artificial host system after exposure to various K_(ATP)modulators, as assayed by abdominal fluorescence.

FIG. 13 depicts the ability of mosquitoes to consume blood followingapplication of vehicle or pinacidil to the body or to the leg. Not shownare micrographs revealing that, following application of pinacidil, whatlittle blood was consumed was diverted to the mosquito crop rather thanthe usual location in the midgut, thereby presumably limiting theability of the mosquito to transmit viral pathogens.

FIGS. 14(A) and (B) depict measurements of aphid (Aphis gossypi) feedingon cotton leaves, as measured through the electrophenegraph technique;on control leaves and on leaves treated with the Kir inhibitor barium,respectively. A total of 8 replicates were studied for control, forbarium-treated leaves, and for VU041-treated leaves. All animals in eachgroup showed a nearly identical response. The animals on the control andVU937 (inactive analog of VU041) leaves fed normally, showing distinctegestion, salivation, and ingestion stages. The animals on the leavestreated with Kir modulators showed no discernable egestion, salivation,or ingestion.

FIGS. 15(A), (B), and (C) depict molecules found to inhibit insect Kirchannels in a thallium flux fluorescent assay, as well as in patch clampelectrophysiology studies.

FIG. 16(A) depicts the molecular structure of pinacidil,(±)-N-Cyano-N′-4-pyridinyl-N″-(1,2,2-trimethylpropyl)guanidine, acyanoguanidine drug that opens ATP-sensitive potassium channels.

FIG. 16(B) depicts total saliva secretion measured in a modified Ramsayassay in the tick Amblyomma americanum, in the presence of dopamine orpinacidil. These data and the data in FIGS. 16(C) and (D) show thatATP-gated Kir channels are critical for proper salivation in the tick.

FIG. 16(C) shows the effect of extrinsic ATP on inhibition of salivationby pinacidil. Secreted salivary volume at 20 minutes is shown in thepresence of dopamine, ATP, pinacidil, or pinacidil+ ATP. Differentletters indicate statistically significant difference (p<0.05).

FIG. 16(D) depicts the concentration dependence of pinacidil-medicatedinhibition after 10 minutes, 20 minutes, and 30 minutes.

FIG. 17(A) depicts the effect of different treatments (no blood meal,blood-fed control, pinacidil, VU041) on the average mass of the mosquitoAedes aegypti following a blood meal. Different letters indicatestatistically significant difference (p<0.05).

FIG. 17(B) depicts the effect of different treatments (control,pinacidil, inactive analog) on the volume of cumulative blood ingestionas a function of time in the tick Amblyomma americanum.

FIG. 18 depicts the structures of VU063 (also called VU0071063), VU625,and an inactive analog of VU063

FIG. 19 depicts mortality of ticks as a function of time for A.americanum for controls, and for ticks treated with pinacidil. Anearly-identical pattern of mortality was observed when ticks wereinstead exposed to VU063 while feeding (data not shown).

MODES FOR PRACTICING THE INVENTION

Ticks.

We used different, structurally distinct, small-molecule modulators todemonstrate the effect of Kir and K2P channels on fluid secretion(salivation) and ion secretion (osmoregulation) in the tick salivarygland. Either barium chloride or VU041, both of which are Kir channelblockers, reduced salivary output by approximately 60%. Bothsignificantly reduced K⁺ and Na⁺ ion secretion rates in salivarydroplets. We also found that the subfamily of Kir channels known asATP-gated potassium (Katp) channels play a critical role in salivation.Pinacidil, an activator of Katp channels, reduced saliva secretion inAmblyomma americanum by 97% as compared to control. Pinacidil'sinhibition of salivation was concentration-dependent, with IC₅₀ valuesof 320 μM, 250 μM, and 120 μM at 10-, 20-, and 30-min of salivation,respectively. We used two tests to verify that pinacidil reducedsalivation through K_(ATP) channel modulation and not by off-targeteffects. First, since K_(ATP) channels are blocked by ATP, and pinacidilis an activator of K_(ATP) channels, we should be able to negate thepinacidil-induced inhibition of salivation by irreversibly blocking thechannel with pre-exposure to ATP. Exposing the salivary glands to 600 μMpinacidil reduced total salivation volume from a control of 150 nL/5 minto 40 nU5 min. However, pre-exposure to ATP negated this effect, with atotal salivation volume of 142 nL/5 min, not statistically significantfrom control.

A second structurally diverse activator, VU063, eliminated salivation inthe isolated salivary gland. Concentration-response curves with VU063showed that VU063 inhibited salivation in isolated tick salivary glandswith an IC₅₀ value of 772 nM, 560 nM, and 410 nM at 10-, 20-, and 30-minof salivation, respectively. Pre-exposure of the gland to ATP negatedthe inhibitory effect of VU063, and the use of an inactive molecule (ananalog known to be inactive in mammalian K_(ATP) channels) did notinfluence salivation. Our data showed that the K2P channels, whichprovide a small outward and/or inward current in excretory cells, play acritical role. In isolated salivary glands fluoxetine, an inhibitor ofK2P channels, reduced salivation by 98%. These data show the criticalimportance of potassium conductance pathways for proper salivary glandfunction, salivation, and osmoregulation, in ticks and other arthropods.Targeting these pathways allows one to control salivation and feedingbehavior. Preventing tick feeding by inhibiting the salivary gland willreduce vector disease transmission, because ticks have a prolongedperiod of feeding, and ticks typically do not begin transmittingpathogens to the host for 18-35 hours.

Modulation of tick Kir/KATP channels dramatically reduced the volume ofblood ingested, and dramatically altered blood feeding biology. Westudied the effects of including pinacidil or VU063 in the blood meal onthe per-tick volume of blood ingested. All ticks were observedattempting to feed by including rhodamine B, a fluorescent tracer, inthe blood meal. Exposure to 400 μM pinacidil reduced the volume of bloodingested by up to 15-fold throughout the course of feeding. Forinstance, at day 6 of feeding, control ticks ingested a volume of 16±4μL per day, while pinacidil-exposed ticks ingested 1.2±0.3 μL per day.Similarly, inclusion of VU063 at a concentration of 100 μM reduced thevolume of ingested blood below the limits of detection; and these ticks'body weights were not statistically different from that of unfed ticks.The inactive analogs to these molecules resulted in ingested bloodvolume similar to that of controls.

Kir channel modulation also altered the tick behavior during bloodfeeding, in a manner that should reduce pathogen transmission. Duringblood feeding, ticks cement themselves onto the host and are capable ofblood feeding for an extended period of time. Once a tick has attachedit will generally not detach on its own accord until a blood meal iscompleted. Control ticks in our system mimicked this behavior. However,including pinacidil or VU063 in the blood meal dramatically altered tickfeeding biology and behavior, as these ticks had a higher rate ofdetachment during blood feeding. Control ticks were found to detach anaverage of 0.11 times per feeding, while pinacidil- and VU063-treatedticks detached an average of 2.1 and 3.6 times per feeding,respectively. This behavior was likely due to failed salivary glands,inadequate feeding, and relocating in an attempt to find a more amenablesite for acquiring blood.

The agent responsible for Lyme disease, the bacterium Borreliaburgdorferi, infects white-footed mice; the vector responsible for Lymedisease, the tick Ixodes scapularis, maintains the reservoir cycle inthe rodents and also transmits the pathogen to humans, dogs, and othermammals. Wide-area campaigns are thought to offer the best prospects forinterrupting the transmission of zoonotic pathogens. Two approaches havebeen tried for controlling Lyme disease transmission control, bytargeting ticks on rodents. One approach uses topical treatments such asfipronil or permethrin administered in bait boxes (e.g., SELECT TCS™Tick Control System) or treated tubes (e.g., Damminix Tick Tubes™). Theother approach uses an oral bait to immunize mice against B.burgdorferi. Our research offers a new approach for interrupting thetransmission of zoonotic pathogens such as B. burgdorferi.Small-molecule modulators that inhibit ion transport in the ticksalivary gland can be used to prevent feeding and to mitigate diseasetransmission. Tick-selective compounds can be incorporated into clothing(e.g. military uniforms, outdoor clothing, etc) to induce salivary glandfailure. This method of fabric impregnation is similar to that currentlyused to kill biting flies with pyrethroids (e.g., Buzz Off™ clothing).

We employed the isolated tick salivary gland to determine the influenceof slight structural variations of pinacidil, acyanoguanidine-containing molecule, on salivary gland function andsaliva secretion. Molecules containing a thiourea, cyanoguanidine, ornitroethene diamines significantly reduced tick salivation, by 80% ormore, at a concentration of 500 μM. Urea-containing molecules had noinfluence on salivation, consistent with previous findings that theyhave no activity on the mammalian KATP channel. Our findings stronglysuggest that: (1) inhibition of salivation is due to pharmacologicalmodulation of KATP channels in the tick salivary gland, and (2) otherKATP channel modulators, besides pinacidil, can also inhibit salivation.

Tick Osmoregulation:

Ticks employ their salivary glands as the tissue responsible forosmoregulation and therefore, inducing salivary gland failure wouldprevent the excretion of salts obtained from the host blood meal. Salivacollections from the isolated tick salivary gland after exposure topinacidil, VU063, or nicorandil showed a significant reduction ofcations (K+ and Na+) concentrations excreted from the salivary glands.These data show that inducing salivary gland failure through KATPchannel inhibition results in altered ion secretion rates and failure toosmoregulate.

Tick Toxicity:

A failure to osmoregulate during blood feeding would result in a buildupof ions at toxic concentrations into the hemocoel. Indeed, incorporationof pinacidil into the blood meal induced a mortality rate of 65%, 72%,88%, 94%, and 99% at days 1-, 2-, 3-, 4-, and 5. Importantly, injectionof pinacidil into a non-blood fed tick was non toxic and injection of K+ions was lethal. These data highlight the notion that 1) KATP channelsare a novel insecticide target site, and 2) inducing salivary glandfailure is a novel mechanism of toxicity.

Mosquitoes.

We identified the proteins Kir2A and Kir3 in the salivary glands ofAedes aegypti mosquitoes. Without wishing to be bound by thishypothesis, and informed by our data from Drosophila, we expect that theKir2A protein is likely an obligate heteromer for the Katp channel inmosquitoes. We investigated the influence of Katp modulators on mosquitosalivation and blood feeding. We employed an established assay tocollect the saliva from a live mosquito. Injection of pinacidil or VU063reduced the secreted saliva volume from 1.4 μL in control animals to 0.3μL or 0.1 μL, respectively. These data suggest that Kir channels arecritical for salivary gland function of mosquitoes.

To determine the role of salivary gland Kir channels in mosquitofeeding, we exposed mosquitoes to pinacidil, VU063, or tolbutamide byadding the respective compounds to blood. Pinacidil, diazoxide, andVU063 are activators of ATP-gated Kir channels. Tolbutamide andglybenclamide are inhibitors of ATP-gated Kir channels. Exposure tothese molecules reduced the total volume of blood consumed by 99%. Theobserved reduction in blood feeding was presumably due to 1) a failingsalivary gland, or 2) inhibition of gustatory processes, or 3) acombination of both. Interestingly, the small amount of blood that wasconsumed was not directed to the mosquito midgut, as is usual duringblood feeding, but instead was diverted to the crop, which is typicallyused for sugar storage after feeding on plant sap. In subsequentexperiments we found that a Katp modulator (pinacidil) inhibitedinfection of mosquitoes by the Chickungunya virus after feeding on ablood meal, presumably as the result of diversion of the blood to thecrop. Mosquitoes exposed to a Katp modulator did not become infectedwith the virus, and were not able to disseminate the virus to thesalivary glands. Such mosquitoes would presumably not act as a diseasevector capable of transmitting the pathogen. Without wishing to be boundby this hypothesis, this diversion of the blood meal is likely due toKatp-mediated depolarization of the sugar cell in the mouthparts, and isthus a gustatory response. This hypothesis is currently being testedthrough single-cell recordings on the sensilla of mosquito mouthparts.Thus far, electrophysiological recordings of the maxilla gustatorysensilla show that exposure to pinacidil elicits a spike in the sugarand bitter cells.

There are multiple modes the present invention can be used againstmosquitoes. Using Katp channels to activate sugar cells to direct thefinal destination of a blood meal (crop versus midgut) is one approach.Katp modulators can be added to repellent sprays, includingcurrently-marketed mosquito repellent sprays. The population ofDEET-tolerant mosquitoes is increasing worldwide. By activating sugarcells on mosquito tarsi and mouthparts after a mosquito alights on ahost, the mosquito's ability to blood-feed is reduced, and pathogendevelopment within the mosquito is inhibited.

Aphids.

In aphids, a specific Kir channel blocker (barium chloride at 300micromolar) was coated onto leaf tissue, and the feeding behavior of theadult aphid was analyzed through the classical electrophenegraphtechnique. The data clearly showed a significant difference in phloemand xylem feeding ability as compared to control animals, highlighted bythe inability of treated aphids to salivate into the plant to initiatefeeding. These data support our finding that functional Kir channels arerequired for proper feeding in aphids. From observations on the threephases of feeding (salivation, egestion, and ingestion), we concludedthat saliva secretion was altered, and that the salivary gland failedafter exposure to Kir channel blockers.

Our data showed that Kir channels represent a critical conductancepathway in the salivary glands of aphids, a pathway that is required forfeeding. No salivation was observed when the aphids were exposed to theKir channel blocker VU041. Our observations were consistent withsalivary gland failure. Exposure to the inactive analog VU937 did notaffect feeding, and all phases of feeding were identical to controlanimals. Kir channel modulators, for example VU041, may be used tocontrol aphid populations in an agricultural setting. Our data showedthat VU041 essentially completely inhibited egestion, salivation, andingestion in the cotton aphid, Aphis gossypii. The active compoundscould be applied as a foliar spray, or as a composition taken up by aplant systemically. A water-soluble compound potent for the aphid Kirchannel could be taken up by xylem and phloem, so that an aphid could beintoxicated immediately upon probing into the plant tissue. This mode ofadministration shares some similarities with the commonly-used aphidinsecticide imidacloprid. Imidacloprid and other existing agents arecommonly sprayed onto the soil, and then transported by the plant toleaf tissue, to ultimately poison aphids upon feeding.

Characterizing Inward-Rectifying Potassium Channels in the SalivaryGlands of Arthropod Disease Vectors.

Inward-rectifying potassium (Kir) channels are a novel target site forcontrolling arthropod disease vectors. Kir channels constitute acritical conductance pathway that drives the function of the Malpighiantubules. Many arthropods use Malpighian tubules for osmoregulation. Inticks, however, the salivary gland is responsible for bothosmoregulation and pathogen transmission. Surprisingly; we have foundthat Kir channels play similar physiological roles in the salivary glandand in Malpighian tubules. These discoveries allow us to target Kirchannels in arthropod salivary glands for vector control.

We used a combination of genetic, pharmacological, physiological, andtoxicological methods to characterize Kir channels in both fly and ticksalivary glands. mRNA expression of Kir channel components is highlyupregulated in arthropod salivary glands, suggesting that Kir channelsconstitute a critical conductance pathway in the salivary glands.

Identification and Physiological Characterization of Inward RectifyingPotassium Channels in the Arthropod Salivary Gland.

The tick salivary gland (SG) is responsible for blood meal acquisition,pathogen dissemination, and osmoregulation. The machinery underlying SGfunction in arthropods is not well-understood. We performed preliminaryfeeding studies in Drosophila melanogaster to assess the effect of Kirchannel inhibition on sucrose consumption. Preliminary data suggestedthat pharmacological inhibition of Kir channels significantly (P<0.0001)reduced the total volume of sucrose solution ingested by individualflies from 1.4 μL/fly to 0.3 μL/fly.

Genetic knockdown of SG-specific Kir channels increased the timerequired to obtain a complete meal by approximately threefold,presumably due to impaired SG function in adult Drosophila. The openreading frames of SG-specific Kir channel constructs for two tickspecies, Ixodes scapularis and Dermacentor variabilis, are being cloned.

Preliminary studies on the isolated tick salivary gland showed thatexposure to barium, a specific Kir channel blocker, significantly(P<0.01) decreased transepithelial fluid secretion from 270 nl/5 min to141 nl/5 min, suggesting that Kir channels constitute a critical K⁺conductance pathway for proper salivary gland function in ticks.

Role of Inward-Rectifying Potassium Channels on Salivary Gland Functionand on Sugar Feeding of the Fruit Fly Drosophila melanogaster.

The arthropod salivary gland plays a critical role in transmittingpathogens. The published literature lacks a detailed understanding ofthe ion conductance pathways responsible for saliva production andexcretion. A superfamily of potassium ion channels known asinward-rectifying potassium (Kir) channels is overexpressed in theDrosophila salivary gland by 32-fold as compared to the whole-body mRNAtranscripts. Our data support the hypothesis that pharmacological orgenetic depletion of salivary gland-specific Kir channels would alterthe efficiency of the salivary gland, and would reduce feedingcapabilities. We used the fruit fly Drosophila melanogaster as a modelorganism for predicting effects in arthropod disease vectors. ExposingD. melanogaster to VU041, a selective Kir channel blocker, reduced thevolume of sucrose consumption by up to 3.2-fold in aconcentration-dependent manner, with an EC₅₀ of 68 μM. An inactiveanalog of VU041, VU937, did not influence feeding, suggesting that thereduced feeding seen with VU041 was indeed due to Kir channelinhibition.

We specifically knocked down Kin in the D. melanogaster salivary glandto assess the role of these channels in the salivary gland. Thegenetically-depleted fruit flies reduced total volume of sucrosesolution ingested, and increased the time spent feeding, bothobservations consistent with reduced salivary gland function. Weobserved what appeared to be a compensatory mechanism at day 1 ofRNAi-treated fruit flies—likely the Na⁺—K⁺-2Cl⁻ cotransporter, or aNa⁺—K⁺-ATPase pump, either of which could help supplement the inwardflow of K⁺ ions, highlighting the functional redundancy in ion fluxcontrol in the salivary glands. This redundancy should not present anyissues for deploying the invention against arthropod pests, becausepharmacological exposure should induce acute poisoning before geneticupregulation of any compensatory mechanisms would have time to takeeffect. This general phenomenon is seen in the fast toxicity thatfollows poisoning with many commercial insecticides. Our findings showedthat Kir channels are a principal conductance pathway in the Drosophilasalivary gland, and constitute a pathway required for sucrose feeding.

The Drosophila salivary gland mainly comprises secretory cells thatsynthesize and secrete proteins required for feeding. The highexpression of Kin in the salivary gland supports our finding that Kirchannels play an important role in promoting salivary secretion.

We have discovered that Kir channels are essential to proper salivarygland function in D. melanogaster, and that the Kir channels arecritical in the highly intricate physiological processes of feeding. Wehypothesize that the same is true in blood-feeding and sap-feedinginsects and arachnids as well. In a set of preliminary experiments, weused pharmacological inhibition and salivary gland-specific geneticdepletion of Kir channels in the model organism D. melanogaster todemonstrate the physiological importance of Kir channels in fly salivarygland function, as measured by sucrose feeding efficiency.

Drosophila stocks and rearing conditions. Four strains of D.melanogaster were used: The wild type Oregon-R (OR) strain was providedby Dr. Jeffrey Bloomquist of the University of Florida, originallydonated by Doug Knipple, Cornell University, Ithaca N.Y., USA. Differentstrains of GAL4-UAS flies were purchased from Bloomington DrosophilaStock Center (Bloomington, Ind., USA). The GAL4-UAS strain 6870 has apromoter that causes constitutive expression in larval and adultsalivary glands of dsRNA for RNAi against Kin (Ir) under upstreamactivating sequence (UAS) control in F₁ hybrids. The strain 41554expresses hairpin RNA (hpRNA) under the control of UAS for RNAi of GFP,and was used as a negative knockdown control. The genotypes of eachstrain are as follows: 6870, w[1118], P{w[+mC]=Sgs3-GAL4.PD}TP1, 42644,y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMS02480}attP2, 41554, y[1]sc[*] v[1]; P{y[+t7.7] v[+t1.8]=VALIUM20-EGFP.shRNA.2}attP2.

All fly strains were maintained in culture at Louisiana StateUniversity. All fly strains were reared on standard medium in Drosophilatubes at 25° C., 12 hour-12 hour photoperiods, and 55% relativehumidity. For dissection, flies were anaesthetized by chilling on ice,and were decapitated before dissecting out salivary glands inSchneider's medium (Invitrogen, Paisley, Scotland, UK).

Reagents.

The Kir channel inhibitor VU041 and the inactive analog VU937 weresynthesized by Dr. Corey Hopkins at the Vanderbilt Center forNeuroscience Drug Discovery using methods described in); D. R. Swale etal., An insecticide resistance-breaking mosquitocide targeting inwardrectifier potassium channels in vectors of Zika virus and Malaria.Scientific Reports vol. 6, article no. 36954 (published online 16 Nov.2016). Dopamine and the D₁/D₂ antagonist fluphenazine dihydrochloridewere purchased from Sigma-Aldrich. FIGS. 1(a) and (b) depict thestructures of VU041 and VU937, respectively.

Feeding Assay.

A capillary feeding assay (CAFE) was used to quantify the volume ofsucrose solution consumed over a period of time; it was performedessentially as described in W. W. Ja, G. B. Carvalho, E. M. Mak, N. N.de la Rosa, A. Y. Fang, J. C. Liong, T. Brummel, S. Benzer, Prandiologyof Drosophila and the CAFE assay. Proceedings of the National Academy ofSciences of the United States of America 104, 8253-8256 (2007);published online EpubMay 15 (10.1073/pnas.0702726104). Both sexes wereused in this assay; nothing in the literature suggested there would beany differential expression between sexes. One adult fly was placed intoa 2 mL glass vial with a screw lid that was pierced with a glassmicrocapillary tube having a truncated 200-0 pipette tip. Themicrocapillary tubes contained 5% (wt/vol) sucrose solution with a 5 μlmineral oil overlay to minimize evaporation. Each experiment included anidentical CAFE chamber without flies to determine evaporative losses(typically 5-10% of the volume), which were subtracted from experimentalreadings. Concentrations of 100 μM dopamine and 100 μM fluphenazine wereused to determine the influence of those agents on fly feeding. See FIG.2A. Concentrations of 200 μM VU041 and 700 μM VU937 (solubility limits)were tested. See FIG. 2B. Exposure to VU041 yielded approximately 30%mortality, while less than 10% mortality was observed in control andVU937-treated animals. All dead flies were excluded from all time pointsof the study regardless of the time of death. Mean (n>25) values areshown in all figure panels, depicting total consumption.

To determine the effect of potassium on the VU041-mediated reduction insugar consumption, 500 μM potassium chloride was added to the 5% sucrosesolution, and ingested volume was determined as described above.Treatments included a negative control of 5% sucrose solution, atreatment control of 5% sucrose solution+500 μM potassium chloride, thetreatment of 5% sucrose solution+500 μM potassium chloride+200 μM VU041,and 5% sucrose solution+200 μM VU041 (FIG. 2D).

A feeding time assay was used to determine how long individual fliesspent on the open end of the capillary tube, presumably feeding on thesugar solution. Flies with genetically-depleted, salivary gland-specificKir channels were used in this experiment, and were monitored 1-, 2-,and 3-days post emergence. A GoPro HERO 3 video camera was mounted infront of the CAFE assay to record the flies over a 24-hour period.Videos were uploaded to a computer. The time spent feeding was measuredand recorded as a mean (n>10) value.

Fluorescence Microscopy.

Individual adult flies were fed 5% sucrose solution plus 500 ppmrhodamine B with the CAFE feeding (described above). Individualspecimens were placed in the well of a glass concave slide, and coveredwith a glass coverslip to prevent air currents in the laboratory frommoving specimens during observation. The slides were placed on the stageof a fluorescence stereomicroscope (SteREO Lumar.V12, Carl Zeiss,Gottingen, Germany) and observed using incandescent illumination.Digital images were captured with AxioVision version 4.6 (Carl Zeiss)with an 800-ms exposure time. The specimens then were observed underfluorescence microscopy using a rhodamine filter cube (excitationwavelength, 540 nm; emission wavelength, 625 nm). Fluorescence imageswere captured at an exposure time of 300 ms. Minimal to noauto-fluorescence of the negative control negated the need to optimizethe fluorescence exposure time.

Genetic Knockdown of Salivary Gland Specific Kir1.

Advances in Drosophila genetics have enabled tissue-specific knockdownof specific genes through the GAL4-UAS system. This technology is basedon the properties of the yeast transcriptional activator Gal4, whichactivates transcription of target genes by binding to an upstreamactivating sequence (UAS). The GAL4-UAS construct binds near the gene ofinterest, which in this case was hairpin RNA (hpRNA) for Kir1, togenetically enhance or decrease mRNA expression. See D. Busson, A. M.Pret, GAL4/UAS targeted gene expression for studying Drosophila Hedgehogsignaling. Methods in molecular biology 397, 161-201(2007)10.1007/978-1-59745-516-9_13); J. A. Fischer, E. Giniger, T.Maniatis, M. Ptashne, GAL4 activates transcription in Drosophila. Nature332, 853-856 (1988); published online EpubApr 28 (10.1038/332853a0); J.B. Duffy, GAL4 system in Drosophila: a fly geneticist's Swiss armyknife. Genesis 34, 1-15 (2002); published online EpubSep-Oct(10.1002/gene.10150). The two components GAL4 and UAS were carried inseparate Drosophila stocks to allow for hundreds of combinatorialpossibilities after a simple parental cross. This study used a strain offly that expressed the GAL4-UAS promoter at all life stages, but only inthe salivary glands, which enabled salivary gland-specific knockdown ofKir1.

Salivary gland-specific knockdown of Kir1 was achieved by crossingvirgin females from a Kir1 RNAi strain (Bloomington stock 42644) withmales from the salivary gland-expressing GAL4-UAS strain (Bloomingtonstock 6870). The flies were given 96 hours to mate and oviposit prior toremoval from the Drosophila tube. F₁ offspring were allowed to emerge,and adults were used in the study immediately upon emergence. Thegenotype of the Kir1 RNAi (Bloomington stock 42644) was located on theX-chromosome, and therefore male GAL4-UAS flies (6870) were crossed withvirgin females from strain 42644 or 41554.

Statistical Analyses.

Concentration response curves and IC₅₀ values using VU041 and VU937 weregenerated by fitting the Hill equation using variable-slope,unconstrained, nonlinear regression analyses performed with GraphPadPrism (GraphPad Software, San Diego, Calif.). Mean cumulativeconsumption values for VU937 and VU041 were compared to daily controlconsumption values by a one-way ANOVA with a Dunns multiple comparisonspost-test. Times spent feeding in Kir1 and GFP genetic knockdown studieswere compared to control values for each day using one-way ANOVA with aDunns multiple comparisons post-test. Statistical significance for allstudies was P<0.05.

RNA isolation, cDNA synthesis, and quantitative PCR. Total RNA wasisolated and extracted from 30 pairs of Drosophila salivary glands usingTRIzol® Reagent (Life Technologies, Carlsbad, Calif.) and purified usingthe RNeasy kit (Qiagen, Valencia, Calif.). First-strand cDNA wassynthesized from poly(A) RNA using the SuperScript® III First-StrandSynthesis System for real-time quantitative PCR (qRT-PCR) (LifeTechnologies) according to manufacturer instructions. qRT-PCR was thenperformed on an Qiagen Rotor Gene Q 2Plex Real-Time PCR System using themanufacturer instructions. Relative quantification was carried out usingthe 2-^(DDCT) method, with beta-actin used as the reference gene.Appropriate controls, such as DNAse and removal of reversetranscriptase, were performed to ensure the sample was not contaminatedwith genomic DNA. All primers were purchased from Life Technologies:primer reference numbers for the irk1 and actin genes were Dm02143600_s1and Dm02361909_s1, respectively. Five biological replicates wereconducted, and each was analyzed in triplicate. FIG. 3 displays theaverage fold-change in mRNA levels relative to that in wild typeOregon-R control salivary glands. The left bar is wild type; the middlebar is GFP-KD (control); and the right bar is irk1-KD. (KD=“knockdown”)

Effect of Pharmacological Inhibition of Kir Channels on Sugar Feeding.

Dopamine is known to stimulate salivation in arthropods, so dopamine wasused as a positive control to determine the utility of the CAFE assayfor measuring alterations in feeding efficacy after exposure to smallmolecules that target salivation pathways. Mean (n>10) daily consumptionvolumes of sucrose for control flies were 1.25 μL 1 day post emergence(PE), 1.75 μL 2 days PE, 2 μL 3 days PE, and 1.35 μL 4 days PE (FIG.2A). Dopamine was found to statistically increase the total volume ofsucrose consumed as compared to control animals. There was a 1.7-foldincrease in consumption at 1 day PE (P<0.05), 1.3-fold at 2 days PE(P<0.05), 1.2-fold at 3 days PE (P<0.05), and 1.4-fold at 4 days PE(P<0.05) (FIG. 2A). The dopamine receptor inhibitor fluphenazinesignificantly reduced total volume of sucrose solution ingested by3.6-fold at 1 day PE (P<0.01), 3.1-fold at 2 days PE (P<0.001), 2.4-foldat 3 days PE (P<0.001), and 2.2-fold at 4 days PE (P<0.001) (FIG. 2A).The ability to measure feeding differences using pharmacological probesof the dopamine receptor enabled exploration of the role of Kir channelsin Drosophila sucrose feeding through small molecules developed againstthe mosquito Kin channel. Mean (n>25) daily consumption of sucrose forcontrol flies during this experiment was 1.3 μL 1 day PE, 1.65 μL 2 daysPE, 1.35 μL 3 days PE, and 1.45 μL 4 days PE, nearly identical to thevalues observed during the dopamine studies. These data are shown inFIG. 2B, and are expressed as cumulative consumption. Pharmacologicalinhibition of Kir channels by the Kir channel inhibitor VU041significantly reduced the total volume of sucrose solution ingested byflies by 2.6-fold at 1 day PE (P: 0.01), 2.7-fold at 2 days PE(P<0.001), 2.9-fold at 3 days PE (P<0.001), and 3.2-fold at 4 days PE(P<0.001) (FIG. 2B). The volume of sucrose solution ingested by fliesexposed to the VU937 inactive analog of VU041 was not significantlydifferent from that of control-treatment flies (FIG. 2B). The influenceof VU041 on feeding was concentration-dependent, with an EC₅₀ of 68 μM(95% CI: 54 μM-79 μM). No difference in consumption was observed withthe inactive analog VU937 at concentrations ranging up to 500 μM (FIG.2C).

To visualize the volume of ingestion of sucrose solution, thefluorophore Rhodamine B was added to the sucrose solution, with orwithout VU041. Micrographs clearly illustrated that exposure to VU041yielded a reduced intensity of fluorescence when compared to controltreated flies, indicative of a reduced volume of sucrose solutioningested, consistent with the data in FIG. 2B.

Effect of Increased Potassium Ions on Total Consumption of Sucrose.

We also hypothesized that Kir channels in Drosophila salivary glands areresponsible for maintaining the high intracellular K⁺ concentration thatprovides the K⁺ ion gradient and enables the outward flow of potassiumions, presumably through Ca²⁺-activated K⁺-channels. To test thishypothesis, the potassium ion concentration in the sucrose solution wasaugmented to increase the potassium equilibrium constant of the channel(E_(k); from the Nernst equation), which indirectly reduces the efficacyof intracellular K⁺ channel inhibitors, such as VU041.

Supplementing the sucrose solution with 500 μM K⁺ did not significantlyalter the total volume of sucrose consumed when compared to the controlanimals (FIG. 2D; open circles, and line immediately underneath the opencircles, respectively). However, increased potassium ion concentrationsignificantly reduced the efficacy of VU041 for all days studied. Themean consumption of flies that were exposed to 500 μM K⁺ and VU041 was1.1 μL, 1.4 μL, 1 μL, and 1.45 μL at days 1-, 2-, 3-, and 4-PE,respectively, which did not differ significantly (P=0.7) from controlflies. Conversely, high significance (P<0.001) was observed when dailyconsumption of sucrose for flies exposed only to VU041 was compared tothat for those exposed to VU041+K⁺. FIG. 2D shows a 2-, 2.3-, 2.3-, and2.75-fold increase in total consumption for days 1-, 2-, 3-, and 4days-PE, respectively.

Knockdown Efficiency of Irk1 in Salivary Glands.

These data show that Kir channels play a critical role in feeding byadult Drosophila. But the possibility that small-molecule inhibitorsmight bind to additional proteins raised the question whether effects ina combination of tissues might be responsible for reducing feedingefficacy. To address this question, Kin mRNA levels were knocked downspecifically in the salivary gland by RNA interference, using theGAL4-UAS system. Data showed the salivary glands of the F₁ progeny ofirk1 knockdown cross expressed 53% less irk1 mRNA relative to the wildtype (OR) and GFP dsRNA knockdown controls (FIG. 3).

Influence of Kir1 Knockdown on Feeding Efficiency.

Pharmacological or genetic depletion of Kir channels inhibits Malpighiantubule function in flies and mosquitoes, which may negatively influencefeeding through an inability to osmoregulate at the level of theMalpighian tubules. Genetic knockdown of salivary gland-specific Kinchannels was employed to confirm that the reduced ingestion of sugarwater shown in FIGS. 2A-D was indeed due to salivary gland failure, andnot reduced osmoregulatory capabilities stemming from tubule failure.The data in FIG. 4A showed a significant reduction in total volumeingested for the Kin knockdown flies at post-emergence day 2 (P: 0.03),day 3 (P: 0.02), and day 4 (P: 0.005) when compared to control, but notat day 1 (P>0.05). The volume of sucrose ingested by the GFP knockdownflies did not differ from control. Although statistical significance wasobserved, a smaller than expected volume difference (c.a. 1 μL) betweencontrol and Kin knockdown flies was observed, perhaps due to the absenceof external stimuli to inhibit continuous feeding within the CAFE assay.The time each individual fly rested on the bottom of the capillary tubewas also assessed—presumably a time when the fly was feeding—at postemergence days 1, 2, and 3. Similar to the total consumption values, nosignificant difference in time spent feeding was observed for day 1, buta significant increase (P<0.001) in time spent feeding was observed forthe Kin knockdown flies over control flies for days 2 and 3, with a 2.3-and 1.9-fold increase, respectively (FIG. 4B).

Discussion.

Despite the critical role the arthropod salivary gland serves inhorizontal transmission of pathogens, an understanding of the machineryrequired for proper gland function is limited. Pharmacological studiesagainst the isolated tick salivary gland have implicated severalcomponents involved in salivary secretion: dopaminergic pathway,Na⁺—K⁺-ATPase, GABA, and the muscarinic acetylcholine receptor. Thepresent results provide compelling data that a superfamily of potassiumion channels, known as inward rectifier potassium channels, is also anessential conductance pathway in the salivary gland that mediates properfeeding in the model organism Drosophila melanogaster. We expect thesechannels to play a similar role in feeding by other arthropods,including mosquitoes, ticks, and aphids.

Insect Kir channels serve a critical role in Malpighian tubule functionand fluid secretion. The Malpighian tubules and salivary glands arephysiologically related tissues: Both are polarized epithelial tissues,both play primary roles in transporting water and ions, and both areconsidered, at least in part, to be exocrine tissues. Furthermore, theKin channel has been shown to constitute the primary inward K⁺conductance channel in the mosquito Malpighian tubule; the homologousgene that encodes Kin in Drosophila is highly upregulated in thesalivary glands of larval and adult flies. We hypothesized that Kirchannels serve a critical role in salivary gland function. We elucidatedthe role of these channels through pharmacological and geneticmanipulations of the Kin channel, measured through feeding efficiency.

We used the recently discovered insect Kir channel modulator (VU041) andan inactive analog (VU937) to characterize the influence of thesemolecules in the feeding cascade. Exposure to VU041 during feedingsignificantly reduced the volume of sucrose ingested, whereas VU937 hadno influence on feeding efficiency, suggesting the observed phenotypeacts through Kir inhibition. However, due to the possibility that smallmolecules might inhibit unintended target sites, and the fact that Kirchannels are highly expressed in the Malpighian tubules, theseobservations alone did not necessarily mean that the observed effect onfeeding was directly due to salivary gland failure. To confirm thathypothesis we also studied salivary gland-specific RNAi-mediatedknockdown of the Kin encoding gene. Results from this genetic depletionof Kin showed a significantly less efficient salivary gland; and, whencombined with the VU041-mediated reduction in sucrose consumption,strongly supported our hypothesis that the Drosophila salivary glandrelies on inward conductance of K⁺ ions through Kir channels.

The data from this study raised the question of the physiological roleof the Kir channels in salivary gland function at the cellular level.Electrolyte secretion in mammalian salivary glands is based on secondaryactive transport of anions, principally Cl⁻ (and/or HCO₃ ⁻) ions. K⁺channels in the basolateral membrane of acinar cells maintain themembrane potential of the apical cell membrane to be more negative thanthe Nernst potential for anions, thereby providing a driving force forsustained electrogenic anion efflux across the apical membrane. Thesecond model for a role for Kir channels in the mammalian salivary glandwas described through cell-attached patch and whole-cell patch-clampstudies. Here, researchers demonstrated the presence of four primary K⁺channels, two of which are the outward mediated Ca²⁺-activated K⁺channel and a Kir channel. The inwardly rectifying property of the Kirchannel was hypothesized to perform fast uptake of accumulated K⁺ ions,in concert with Na⁺—K⁺ ATPase, into acinar cells with the K⁺ influxdepending on the relation between the membrane potential and theconcentration gradient of K⁺ across the basolateral membrane. Thisbuffering action likely provides an ion gradient enabling the outwardflow of K⁺ ions through Ca²⁺-activated K⁺ channels.

Our experiments elucidated the role of Kir channels in the insectsalivary gland. The potassium ion concentration in the sucrose solutionwas augmented to increase the potassium equilibrium constant of thechannel (E_(k); from the Nernst equation), which ultimately reduces theefficacy of intracellular K⁺ channel inhibitors, such as Kir channelblockers. The loss of VU041 potency supported the notion that Kirchannels provide a pathway for rapid influx of K⁺ ions afterdepolarization events, a phenomenon often referred to as K⁺-spatialbuffering. It was hypothesized that Kir channels in Drosophila salivaryglands are responsible, at least in part, for maintaining a highintracellular K⁺ concentration through a buffering-like action, whichprovides the K⁺ ion gradient to enable the outward flow of potassiumions, presumably through Ca²⁺ -activated K⁺-channels.

Kir channels are not the only transport pathway facilitating inward flowof K⁺ ions. Genetic depletion of Kir1 channels yielded a reduction offeeding at days 2, 3, and 4, but not on day 1. The time spent feedingwas not statistically different from controls at day 1 when compared tosubsequent days. These data suggest the presence of a compensatorymechanism that accounts for the reduced expression of Kin in thegenetically depleted animals, but that is lost after day 1. Compensatorymechanisms are commonly observed in animals with genetic depletions ofKir channels, and may arise through upregulation of a different Kirgene. For instance, it has been reported that individual knockdown ofany of the three Kir channel genes in Drosophila Malpighian tubules hasno effect on organ function, yet simultaneous knockdown of irk1 and irk2has significant effects on transepithelial K⁺ transport, suggesting thatKir1 and Kir2 play redundant roles in Malpighian tubule function. Kir1and Kir2 mRNA are both expressed in the Drosophila salivary gland,albeit with dramatically different mRNA expression levels. It isplausible that the Ir2 gene is upregulated after genetic depletion ofKir1, which may account for the absence of an effect on feeding atday 1. Furthermore, the Malpighian tubules partially rely on theNa⁺—K⁺-2Cl⁻ cotransporter and Na⁺—K⁺-ATPase pump to establish a highintracellular K⁺ ion gradient. The compensatory systems of theMalpighian tubules and the expression of the same conductance pathwaysin the salivary glands highlight the possibility that the Drosophilasalivary gland is capable of utilizing the same pathways forestablishing the intracellular K⁺ ion concentration as well as providingredundancy in the system for salivary gland K⁺ excretion. These resultsprovide proof-of-concept that VU041 can be used as part of a vectorcontrol agent for disrupting blood feeding and pathogen transport.

An Insecticide Resistance-Breaking Mosquitocide Targeting InwardRectifier Potassium Channels in Vectors of Zika Virus and Malaria.

We have discovered a topically active, small-molecule mosquitocide(VU041) with a novel mechanism of action; viz., inhibition of inwardrectifier potassium (Kir) channels. VU041 is equally toxic againstrepresentative insecticide-susceptible and insecticide-resistant strainsof Aedes aegypti and Anopheles gambiae. However, it is non-toxic tohoney bees. Topical application of VU041 inhibits post-blood-mealfluid-volume regulation. Lead optimization efforts yielded a VU041derivative, named VU730, which is topically active and highly selectivefor mosquito versus mammalian Kir channels. VU041 and its active analogscan be used as a safe and selective mosquitocide to combat the emergingproblem of insecticide-resistance.

VU041 is a submicromolar-affinity inhibitor of Anopheles (An.) gambiaeand Aedes (Ae.) aegypti Kir1 channels. VU041 incapacitates adult femalemosquitoes from representative insecticide-susceptible and -resistantstrains of An. gambiae (G3 and Akron, respectively) and Ae. aegypti(Liverpool and Puerto Rico, respectively) following topical application.VU041 is selective for mosquito Kir channels over several mammalianorthologs, with the exception of Kir2.1. VU041 is not lethal to honeybees. An analog, VU730, retains activity toward mosquito Kir1 but is notactive against Kir2.1 or other mammalian Kir channels. Thus, VU041 andVU730 and their analogs can be used as new classes of insecticides tocombat insecticide-resistant mosquitoes and the transmission ofmosquito-borne diseases, such as Zika virus, without harmful effects onhumans, other mammals, or beneficial insects.

We have discovered that inward rectifier potassium (Kir) channels can beused as targets for new mosquitocides. In Drosophila melanogaster,embryonic depletion of Kir1, Kir2, or Kir3 mRNA levels leads to death ordefects in wing development. Knocking down Kir1 and Kir2 mRNA expressionin the heart and Malpighian (renal) tubules of Drosophila, respectively,inhibits the immune response against cardiotropic viruses andtransepithelial secretion of fluid and K⁺. In yellow fever mosquitoes(i.e. Ae. aegypti), pharmacological inhibition of Kir1 withstructurally-distinct small molecules (e.g., VU573, VU590, or VU625)disrupts the secretion of fluid and K⁺ in isolated Malpighian tubules,and impairs flight, urine production, and K⁺ homeostasis in intactfemales. It is desirable to have inhibitors that are specific formosquito Kir channels over mammalian Kir channels, since the latter playfundamental roles in nerve, muscle, endocrine, and epithelial cellfunction in humans and other mammals.

We have discovered that VU041, a Kir1 inhibitor, exhibits similartoxicity to adult female mosquitoes from representativeinsecticide-susceptible and -resistant strains of An. gambiae (G3 andAkron, respectively) and Ae. aegypti (Liverpool and Puerto Rico,respectively). Moreover, topical VU041 application to adult femalemosquitoes of both species inhibits their fecundity. Importantly, VU041is selective for mosquito Kir channels over mammalian Kir channelorthologs, and it is non-lethal to adult honey bees (Apis mellifera).VU041 may be used to control mosquitoes without harmful effects onhumans and beneficial insects.

VU041.

VU041 is the molecule1-(3,4-dihydroquinolin-1(2H)-yl)-2-(3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)ethan-1-one.VU041 is a potent inhibitor of AnKir1-dependent thallium (Tl⁺) flux invitro. VU041 has a high partition coefficient (c Log P>4) forpenetrating the mosquito cuticle.

In whole-cell patch clamp experiments, VU041 inhibited AnKir1 with anIC₅₀ of 496 nM (95% CI: 396-619 nM; Hill coefficient value of 1.3),making it the second most potent in vitro inhibitor of mosquito Kinchannels discovered to date. (VU625 is a more potent in vitro inhibitor(IC₅₀˜100 nM), but it is not topically toxic to mosquitoes, whichprevents practical use.)

The selectivity of VU041 for mosquito vs. mammalian Kir channels wasevaluated in quantitative Tl⁺ flux experiments against AnKir1, AeKir1,and a panel of Kir channels that play critical physiological roles inmammals: Kir1.1 (kidney), Kir2.1 (heart, brain), Kir4.1 (kidney, brain),Kir6.2/SUR1 (pancreas, brain), and Kir7.1 (broadly expressed). VU041inhibited AnKir1 and AeKir1 with IC₅₀ values of 2.5 μM and 1.7 μM,respectively. VU041 inhibited mammalian Kir1.1, Kir4.1, Kir6.2/SUR1, andKir7.1 by less than 10% at a concentration of 30 μM. The only mammalianKir channel tested that VU041 inhibited appreciably was Kir2.1 (IC₅₀ of12.7 μM).

Analogs of VU041 to Enhance Potency and Mosquito Selectivity.

Analogs of VU041 were synthesized with the goal of improving theselectivity of a VU041-like molecule for AnKir1 vs. Kir2.1. The firstlibrary of analogs was designed to keep the dihydroquinoline portion ofthe molecule constant, evaluating variations in the heterocyclicportion. One compound (VU730, FIG. 5) retained activity toward AnKir1(1050=2.4 μM in Tl⁺ flux assays; IC₅₀=717 nM in patch clamp experiments,but lost activity toward Kir2.1 (IC₅₀>30 μM in Tl⁺ flux assays). Thenext library of analogs kept the trifluoromethyl tetrahydropyrazoleportion of the molecule constant, while varying the amide portion of themolecule. Although none of the compounds in this series showed anincrease in potency against AnKir1, VU937 (FIG. 5) inhibited AnKir1channel activity in patch clamp experiments by 60-fold less than VU041(IC₅₀=29.7 μM; 95% Cl: 17.7-49.9 μM). With its significantly lowerpotency, VU937 was used in subsequent experiments as an ‘inactive’analog to confirm that any toxic or physiological effects of VU041 onmosquitoes were associated with the inhibition of Kir1.

VU041 is Equally Toxic to Insecticide-Susceptible and -Resistant Strainsof Mosquitoes.

A distinct advantage of the present invention is that it is effectiveagainst mosquito populations that have developed resistance toconventional insecticides.

To determine the topical toxicity of VU041 against mosquitoes, thecompound was applied to the cuticles of insecticide-susceptible andinsecticide-resistant strains of An. gambiae and Ae. aegypti (adultfemales), and efficacy was assessed 24 h later. The resistant ‘Akron’strain of An. gambiae is resistant to permethrin (33-fold) and propoxur(101-fold) when compared to the susceptible G3 strain of An. Gambiae.The ‘Akron’ strain is known to express resistance through target-site(kdr) and Modified Acetylcholine Esterase (MACE) and metabolicresistance mechanisms. The resistant ‘Puerto Rico’ (PR) strain of Ae.aegypti possesses target-site (kdr) resistance (J. J. Becnel and BEIresources, personal communications), which contrasts with another PuertoRican strain that possesses elevated mRNA levels encoding CYP450enzymes. The ED₅₀, or effective dose to incapacitate 50% of themosquitoes, for VU041 was similar for the insecticide-susceptible and-resistant strains of each species (FIGS. 6(A) & 6(B)). In both species,VU937 was not toxic, suggesting that the toxicity of VU041 wasassociated with its inhibition of Kin channels.

Pretreatment of the susceptible (G3) strain with piperonyl butoxide(PBO), an inhibitor of cytochrome P450 monoxygenases (CYP450s), enhancedthe efficacy of VU041 by ˜3-fold, whereas pre-treatment withS,S,S-tributyl phosphorotrithioate (DEF), an inhibitor ofcarboxylesterases, did not enhance toxicity. Inhibition of CYP450s inthe AKRON strain, which overexpress some CYP450 genes up to 12-fold,enhanced toxicity 3-fold over the G3 strain, likely due to the increasedlevels of metabolic enzymes and the resulting altered pharmacokineticsand pharmacodynamics in the resistant strain. Thus VU041 was onlymoderately metabolized by cytochrome P450 enzymes, and it did not appearto be metabolized by esterases. Experiments in the G3 strain of An.gambiae with VU730, which does not inhibit mammalian Kir2.1, showed anED₅₀ similar to that for VU041. Thus, VU041 is believed to be the firstreported small-molecule inhibitor of mosquito Kin channels that exhibitstopical toxicity against both insecticide-susceptible andinsecticide-resistant lines of mosquitoes. Moreover, the structure VU041can be modified to reduce its inhibition effects against mammalianKir2.1 without affecting its efficacy as a mosquitocide (e.g., VU730).

VU041 Inhibits Renal Excretory Function in Mosquitoes.

A signature feature of inhibiting Kir channels in mosquitoes isimpairment of fluid secretion/urine production in Malpighian tubules,which reduces the mosquito's diuresis. Diuresis plays an especiallyimportant role in adult female mosquitoes after a blood meal; excessfluid and electrolytes are excreted and absorbed into the hemolymph.Experiments were conducted to determine whether VU041 disruptsfluid-volume regulation associated with blood meal processing in An.gambiae mosquitoes. Immediately after engorgement, mosquitoes weretreated with an ED₃₀ dose of VU041, and their abdominal diameters weremeasured over the following 24 h. In vehicle (control)- andVU937-treated mosquitoes, abdominal diameter increased approximately2-fold immediately following blood feeding (FIG. 6(C)), and thendecreased significantly over 24 h. In striking contrast, although theabdominal diameter of VU041-treated mosquitoes initially increasedsimilarly, it did not thereafter change much during the following 24hours, an observation that is consistent with VU041-dependent inhibitionof fluid-volume excretion.

To directly determine whether VU041 impairs mosquito excretion, an invivo diuresis assay was performed on adult female Ae. aegypti, with theinhibitors applied topically. The diuretic capacities of control andVU937-treated mosquitoes were similar to one another, whereas thediuretic capacity of VU041-treated mosquitoes was significantly lower,by ˜51%, as compared to controls (FIG. 6(D)). Taken together with thedata in FIG. 6(C), these results suggest that VU041 impairs renalexcretory function and fluid-volume regulation during blood mealprocessing in mosquitoes.

VU041 Reduces Mosquito Fecundity.

VU041 disrupts blood meal processing and diuresis in mosquitoes (FIG.6(C) & 6(D)). Knockdown of AnKir1 expression via RNA interferencereduces fecundity. We hypothesized that VU041 would also reduce egglaying after blood feeding. Adult female mosquitoes of both species weretopically treated with ˜1 μg/mg mosquito (An. gambiae) or 3.4 μg/mgmosquito (Ae. aegypti) of VU041 or up to ˜10 μg/mg mosquito (solubilitylimit) of VU937 within 1 h after engorgement, and the total number ofeggs laid per mosquito was counted 72 h post blood feeding. For both An.gambiae and Ae. aegypti, the control and VU937-treated mosquitoes laid asimilar median number of eggs per mosquito, whereas the VU041-treatedmosquitoes laid a significantly lower median number of eggs per mosquito(FIGS. 7A & 7B). Thus, VU041 reduced mosquito fecundity.

VU041 is not lethal to adult honeybees.

Insecticidal activity against pollinators is undesirable, a factor whoseimportance has been highlighted in recent discussions about the role ofinsecticides in the decline of pollinator health. To determine whetherVU041 is toxic to honey bees, 3-day old adult honey bees were treatedtopically on the thoracic notum with a limit dose of VU041 (1 mg/bee;i.e., ˜10 μg/mg). Toxicity was assessed 48 h later compared to negative(vehicle) and positive (0.1 μg/bee bifenthrin) controls. As shown inFIG. 8, VU041 did not cause significant mortality to honey bees within48 h as compared to the vehicle (Fisher's Exact Test, P=0.74, N=130),whereas application of bifenthrin resulted in 100% mortality at 48 h(P<0.001, N=87). Thus, VU041 is non-lethal to adult honey bees whenapplied topically.

Discussion.

Our data showed that a small molecule inhibitor of mosquito Kir channels(VU041) is a mosquitocide that overcomes insecticide resistance, andthat it should be safe for humans and insect pollinators.

VU041 Circumvents Existing Insecticide-Resistance Mechanisms.

The Akron strain of An. gambiae used in some of our experiments carriesmultiple resistance mechanisms, including: 1) mutations in avoltage-gated Na⁺-channel (kdr) that imparts resistance to pyrethroids,2) mutations in an AChE (MACE, ace-1R) that confers resistance tocarbamates, and 3) metabolic resistance derived from increased levels ofCYP450s and carboxylesterases. The Puerto Rican (PR) strain of Ae.aegypti used in our experiments is resistant to pyrethroids via a singlemechanism: a point mutation (kdr) in the voltage-gated sodium channel.

VU041 had similar efficacy against the LVP and PR strains of Ae.aegypti. However, mosquito strains with both target-site and metabolicresistance, such as the Akron strain of An. gambiae, might reasonably beexpected to have the capacity to detoxify small molecules generally,regardless of the specific molecular target. However, VU041 showedsimilar efficacy against both the Akron and G3 strains of An. gambiae,showing that the detoxification mechanisms of the resistant Akronmosquitoes were ineffective against this new molecule. Consistent withthis finding, inhibiting CYP450s with PBO, or inhibiting esterases withDEF only nominally enhanced the toxicity of VU041 against the G3 strainof An. gambiae, showing that VU041 is inefficiently metabolized by theseimportant detoxifying enzymes. By contrast, inhibiting CYP450s in theAkron strain enhanced toxicity of VU041 at a greater level as comparedto the G3 strain, presumably due to the significant overexpression ofmultiple CYP450 genes in this resistant strain. These data areconsistent with the hypothesis that VU041 is a “modest” substrate ofthis class of detoxification enzymes.

Mechanisms of Action of VU041 Against Mosquitoes.

In vivo experiments on blood meal processing and diuretic capacitysuggested that one mechanism of action of VU041 is the disruption ofexcretory functions mediated by Malpighian tubules. The inhibition ofKir channels in Malpighian tubules could disrupt the processing of bloodmeals by limiting the excretion of blood-derived electrolytes and waterabsorbed into the hemolymph. There could also be effects on the midgut'sdigestion of blood, or absorption of ions or fluid from the blood. Themosquito midgut is a site of Kir mRNA expression and barium-sensitive K⁺transport. Our experiments in An. gambiae demonstrated that mosquitoestreated with VU041 retained a large abdominal girth 24 h after bloodfeeding while control mosquitoes did not, suggesting that themosquitoes' processing of blood meals had been impaired—perhaps bydisrupting post-prandial diuresis, or perhaps by disrupting blooddigestion. Experiments in Ae. aegypti confirmed that VU041 inhibitedmosquito diuretic capacity, which would be consistent with impairment ofMalpighian tubule function. This mechanism of action, on a targetoutside the nervous system, may also contribute to the efficacy of VU041against the Akron and PR strains, which are resistant to neurotoxiccarbamates and pyrethroids.

The combined effects of VU041 on blood-meal processing and diuresis mayalso contribute to its inhibition of fecundity in the mosquitoes An.gambiae and Ae. aegypti. That is, if VU041 inhibits blood mealdigestion, nutrient absorption, and solute and metabolite excretion,then vitellogenesis may not proceed efficiently. Another possibility isa direct effect on the ovaries. Or there may be a combined effect on theexcretory and reproductive systems.

Selectivity of VU041 for Mosquito Vs. Mammalian Kir Channels.

The selectivity of any new mosquitocide is important, because mosquitocontrol is typically implemented near or even within human dwellings,especially in tropical regions having endemic malaria, dengue, or Zika.Typical modes of administration include, e.g., aerial sprays, orinsecticide-treated bed nets. Our in vitro screening assays for VU041show that this compound has a relatively “clean” ancillary pharmacologyagainst a panel of mammalian Kir channels, with no meaningful activityagainst Kir1.1, Kir4.1, Kir7.1, and Kir6.2/SUR1. However, VU041moderately inhibits mammalian Kir2.1, which is highly expressed in thehuman heart; inhibition of this channel might have deleteriousconsequences on mammalian heart function. We therefore developed andtested analogs of VU041 to identify structural changes promotingincreased selectivity for AnKir1 vs. Kir2.1. The compound VU730 retainedinhibition activity against AnKir1, but it had no measureable inhibitionagainst Kir2.1.

VU730 retained topical mosquitocidal toxicity, with a similar potency tothat of VU041. VU730 is expected to be non-toxic to mammals, as will beconfirmed through routine testing.

Selectivity of VU041 for Mosquitoes Vs. Adult Honey Bees.

VU041 is toxic against mosquitoes, but has minimal effects on beneficialinsects. Ideally, new mosquitocides should also have limited or noeffects on beneficial insects, such as honey bees and other pollinators.Existing, broad-spectrum insecticides (e.g., neonicotinoids,pyrethroids) can adversely affect pollinator health. Remarkably, weobserved that a dose of 10 μg VU041 per mg in adult honey bees (A.mellifera) was not toxic after 48 h. The same dose would be ˜100% lethalagainst mosquitoes within 24 h. The honey bee ortholog of Kin sharesonly ˜55% amino acid identity with mosquito Kin channels. Thus, withoutwishing to be bound by this hypothesis, the interaction of VU041 withmosquito Kin channels may involve a domain that is not conserved withthe bee Kin channel. Alternatively, without wishing to be bound by thishypothesis, the chemical composition of the honey bee cuticle maysubstantially differ from that of mosquitoes in a manner that reducesthe penetration of VU041 into the hemolymph; or the VU041 molecule maybe more efficiently detoxified by bees.

T-REx-HEK293 Cell Line Expressing Kir Channels.

The open-reading frame of a full-length cDNA encoding AnKir1 cloned fromAn. gambiae Malpighian tubules (Genbank Accession # KJ596497) wassub-cloned into the pcDNA5/TO expression vector (Life Technologies) andused for stable cell line generation.

Chemical Synthesis; Tl⁺ Flux Assay.

The thallium flux assay is an in vitro method of measuring conductancethrough a potassium ion channel. Potassium channels are also permeableto thallium ions. The modulation of a K⁺ channel will thus increase ordecrease thallium ion flow through the channel and thus, alter theobserved fluorescence of a thallium-specific indicator dye.

Stably transfected T-Rex-HEK-293-AnKir1 cells were cultured overnight in384-well plates in media containing DMEM, 10% dialyzed FBS, and 1 μg/mLtetracycline to induce channel expression. The next day the cell culturemedium was replaced with a dye-loading solution containing assay buffer(Hanks Balanced Salt Solution with 20 mM HEPES, pH 7.3), 0.01% (w/v)Pluronic F-127 (Life Technologies, Carlsbad, Calif.), and 1.2 μM of thethallium-sensitive dye Thallos-AM (TEFlabs, Austin, Tex.). After 1 hrincubation at room temperature, the dye-loading solution was washed fromthe plates and replaced with 20 μL/well of assay buffer. The plates weretransferred to a Hamamatsu Functional Drug Screening System 6000(FDSS6000; Hamamatsu, Tokyo, Japan), where 20 μL/well of each of thetest compounds in assay buffer was added and allowed to incubate withthe cells for 20 min. After incubation, a baseline recording wascollected at 1 Hz for 10 s (excitation 470±20 nm, emission 540±30 nm); athallium stimulus buffer was then added (10 μL/well), and data were thencollected for an additional 4 min. The Tl⁺ stimulus buffer contained (inmM): 125 NaHCO₃, 1.8 CaSO₄, 1 MgSO₄, 5 glucose, 12 Tl₂SO₄, 10 HEPES, pH7.4. For Tl⁺ flux assays on Kir2.x, Kir4.1 and Kir6.2/SUR1 expressingcells, the Tl⁺ stimulus buffer contained 1.8 mM Tl₂SO₄. To ensure thesmall-molecule vehicle DMSO had no direct effect on AnKir1-dependent Tl⁺flux, the assay's tolerance to different doses of DMSO was evaluated.The robustness and reproducibility of the assay was determined bycomparing Tl⁺ flux through tetracycline-induced and tetracycline-freecells. The Z′ value was calculated as:

Z′=1−(3SDp+3SD_(n))/|mean_(p)+mean_(n)| where SD is standard deviation,and p and n are control and uninduced flux values, respectively. Tocompare the effect of DMSO on AnKir1-mediated Tl⁺ flux, a one-way ANOVAwas performed with a Tukey's multiple comparison test. Prism software(GraphPad Software) was used to generate CRC from Tl⁺ flux.Half-inhibition concentration (IC50) values were calculated from fitsusing a four parameter logistic equation.

High-Throughput Screening.

Test compounds were transferred to daughter, 384-well polypropyleneplates (Greiner Bio-One, Monroe, N.C.) with an Echo555 liquid handler(Labcyte, Sunnyvale, Calif.), and then diluted into assay buffer togenerate a 2× stock in 0.6% DMSO (0.3% final). For Tl⁺ flux assays onKir6.2/SUR1 expressing cells, test compounds were diluted in assaybuffer containing diazoxide (250 μM final) to induce channel activation.Concentration-response curves (CRCs) were generated by screeningcompounds at 3-fold dilution series (1 nM-30 μM).

Tl⁺ flux data were analyzed using Excel (Microsoft Corp, Redmond, Wash.)with XLfit add-in (IDBS, Guildford, Surrey, UK), OriginPro (Origin Lab,Northampton, Mass.), and GraphPad Prism (GraphPad Software, San Diego,Calif., USA) software. Raw data were opened in Excel and each data pointin a given trace was divided by the first data point from that trace(static ratio) followed by subtraction of data points from controltraces generated with vehicle controls. The slope of the fluorescenceincrease beginning 5 s after Tl⁺ addition and ending 15 s after Tl⁺addition was calculated.

Patch Clamp Electrophysiology.

Patch electrodes were pulled from silanized 1.5 mm outer diameterborosilicate microhematocrit tubes using a P-1000 Flaming/Brownmicropipette puller (Sutter Instrument, Novato Calif., USA). Electroderesistance ranged from 2-4 MΩ. Whole-cell currents were recorded undervoltage-clamp conditions using an Axopatch 200B amplifier (MolecularDevices, Sunnyvale, Calif.). Electrical connections to the amplifierused Ag/AgCl wires and 3 M KCl/agar bridges. Electrophysiological datawere collected at 5 kHz and filtered at 1 kHz. Data acquisition andanalysis were performed using pClamp 9.2 software (Axon Instruments).

Two-Electrode Voltage Clamp Electrophysiology.

Defolliculated Xenopus laevis oocytes (Ecocyte Bioscience, Asutin, Tex.)were injected with AeKir1 or AeKir2B cRNA (10 ng) and cultured in OR3media for 3-7 days at 18° C. before electrophysiology experiments. Whenpresent, VU041 was dissolved in solution III (See Table S4) to a finalconcentration of 50 μM (0.05% DMSO). All solutions were delivered bygravity at a flow rate of ˜2 ml/min to an RC-3Z oocyte chamber (WarnerInstruments, Hamden, Conn.) in polyethylene tubing, and solution changeswere performed with a Rheodyne Teflon 8-way Rotary valve (Model 5012,Rheodyne, Rohnert Park, Calif.).

For each experiment, an oocyte was transferred to the RC-3Z chamberunder superfusion (solution I). To measure membrane potential (V_(m))and whole-cell membrane current (I_(m)) of the oocyte, it was impaledwith two glass microelectrodes backfilled with 3 M KCl (resistances of0.5-1.5 MO). Each microelectrode was bridged to an OC-725 oocyte clamp(Warner Instruments) and was under the digital control of pCLAMPsoftware (Clampex module, version 10, Molecular Devices, Sunnyvale,Calif.).

The voltage clamp was then turned off during solution changes, and whenthe oocyte reached a new steady-state V_(m) (˜90 s) the I-V relationshipof the oocyte was measured again. All V_(m) and I_(m) values weredigitally recorded (Digidata 1440A Data Acquisition System, MolecularDevices) and the resulting I-V plots were generated with the Clampfitmodule of pCLAMP.

To measure the inhibition of Kir channel activity by VU041, we focusedon the maximal inward currents elicited, which occurred at a clampvoltage of −140 mV during the voltage-stepping protocol. The backgroundcurrent of an oocyte in solution II (low K⁺) was subtracted from thatin 1) solution III (elevated K⁺) to calculate the inward current beforeexposure to VU041 (IA), and 2) solution III with VU041 to calculate theinward current after exposure to a small molecule (I_(B)). The percentinhibition of I_(A) by VU041 was calculated by subtracting I_(B) fromI_(A) and then dividing by I_(A).

Chemical Analysis.

All NMR spectra were recorded on a 400 MHz FT-NMR DRX-400 FT-NMRspectrometer or a 500 MHz Bruker DRX-500 FT-NMR spectrometer. ¹Hchemical shifts are reported as δ values in ppm downfield, withdeuterated solvent as the internal standard. Data are reported asfollows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet,q=quartet, br=broad, m=multiplet), integration, coupling constant (Hz).High resolution mass spectra were recorded on a Waters Q-TOF API-US plusAcquity system with electrospray ionization. Reversed-phase LCMSanalysis was performed using an Agilent 1200 system with a binary pumpwith degasser, high-performance autosampler, thermostatted columncompartment, diode-array detector (DAD) and a C18 column. Flow from thecolumn was split to a 6130 SQ mass spectrometer and a Polymer Labs ELSD.The MS detector was configured with an electrospray ionization source.Data acquisition was performed with Agilent Chemstation and AnalyticalStudio Reviewer software. Samples were separated on a ThermoFisherAccucore C18 column (2.6 um, 2.1×30 mm) at 1.5 mL/min, with column andsolvent temperatures maintained at 45° C. The gradient conditions were7% to 95% acetonitrile in water (0.1% TFA) over 1.1 minutes.Low-resolution mass spectra were acquired by scanning from 135 to 700AMU in 0.25 seconds with a step size of 0.1 AMU and peak width of 0.03minutes. Drying gas flow was 11 liters per minute at a temperature of350° C., and a nebulizer pressure of 40 psi. The capillary needlevoltage was 3000 V, and the fragmentor voltage was 100 V. Preparativepurification was performed on a custom HP1100 purification system withcollection triggered by mass detection. Solvents for extraction, washingand chromatography were all HPLC grade. All reagents were purchased fromAldrich Chemical Co. and were used without purification.

General Procedures for Compound Synthesis.

Chloroacetyl chloride (1.3 eq.) was added to a solution of an amine (1eq.) and pyridine (4 eq.) in DMF (2 mL). After 30 min at roomtemperature, the reaction was added to a mixture of EtOAc and water(1:1). The aqueous layer was extracted with EtOAc, and the organicextraction was washed with water. The organic extraction wasconcentrated under reduced pressure to yield the desiredα-chloroacetamide.

A solution of the α-chloroacetamide (1 eq.), an amine (1 eq.) and cesiumcarbonate (1 eq.) in DMF was heated to 150° C. for 5 min in a microwavereactor. The solution was filtered (0.45 μm), and fractions wereseparated via reverse-phase HPLC in a gradient of MeCN in water (0.1%TFA). Fractions were combined and added to water:EtOAc (1:1) and addedaq. NaHCO₃. The organic layer was collected and solvent was removed onan air concentrator. Residue was resuspended in DCM/MeOH and filteredthrough a phase separator into a vial yielding the desired finalproducts.

Synthesis of 2-chloro-1-(3,4-dihydro-2H-quinolin-1-yl)ethanone (1)

See FIG. 9. Chloroacetyl chloride (0.16 mL, 2.0 mmol) was added to asolution of 1,2,3,4-tetrahydroquinoline (0.19 mL, 1.5 mmol) and pyridine(0.50 mL, 6.2 mmol) in DMF (2 mL). After 30 min at room temperature, thereaction was added to a mixture of EtOAc and water (100 mL:100 mL). Theaqueous layer was extracted with EtOAc (100 mL). The organic extractionwas washed with water 3× (300 mL). The organic extraction wasconcentrated under reduced pressure to yield Compound 1 (243 mg, 1.16mmol, 77% yield). LCMS: 0.90 min, >90% at 215 and 254 nm, [M+H]⁺=210.2.

Synthesis of1-(3,4-dihydroquinolin-1(2H)-yl)-2-(3-(trifluoromethyl)-4,5,6,7-tetrahydro-11-1-indazol-1-yl)ethan-1-one(VU041)

See FIG. 9. A solution of Compound 1 (25 mg, 0.12 mmol),3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazole (22.7 mg, 0.12 mmol)and cesium carbonate (38.8 mg, 0.12 mmol) in DMF (1 mL) was heated to150° C. for 5 min in a microwave reactor. The solution was filtered(0.45 μm) and fractions were separated via reverse-phase HPLC in agradient of MeCN in water (0.1% TFA). Fractions were combined and addedto a mixture of water (15 mL) and EtOAc (15 mL). To the mixture wasadded a saturated solution of NaHCO₃(1 mL). The organic layer wascollected and solvent was removed on an air concentrator. The residuewas resuspended in DCM:MeOH and filtered through a phase separator intovials, yielding VU041 (43.4 mg, 0.0523 mmol, 44% yield). LCMS: RT=0.793min, >95% at 215 and 254 nm, [M+H]⁺=364.1.

Lead Compound Optimization.

Commercial 1,2,3,4-tetrahydroquinoline was treated with chloroacetylchloride in the presence of pyridine to yield Compound 1. Next, theappropriate nitrogen heterocycle was reacted in a microwave reactor withCompound 1 under basic conditions, yielding the product compounds. Afirst library was designed to keep the dihydroquinoline moiety constant,and evaluate variations in the heterocyclic portion. If the six-memberedring was aromatized, the compound lost activity against AnKir1, 2.Addition of a carbonyl group to the 4-position of thetetrahydroindazole, Compound 3, also led to an inactive compound.Interestingly, deletion of a nitrogen from Compound 3 brought someactivity back into Compound 4 (12.1 μM). One compound (VU730, Compound5) retained activity toward AnKir1 (IC₅₀=2.4 μM), but lost activitytoward Kir2.1 (IC₅₀>30 μM). Expanding the ring system to incorporate atetrahydroquinoline retained some activity against AnKir1 (Compound 6,8.0 μM), and deletion of the 6-membered ring of thetetrahydroisoquinoline leaving the unsubstituted pyrazole wasunproductive (Compound 7, inactive).

A second library kept the trifluoromethyl tetrahydropyrazole moietyconstant, while varying the amide portion of the molecule. Moving fromthe tetrahydroquinoline to the tetrahydroisoquinoline led to a ˜7-foldloss of potency (Compound 8, 15 μM). Moving to the decahydroquinoline(Compound 9, 6.7 μM) retained some activity while the regioisomer wasinactive (Compound 10). Moving to a piperidine ring was not productive(Compound 11); however, adding pendant substitution led to activecompounds (phenyl, Compound 12, 5.6 μM and dimethyl, Compound 13, 4.5μM). Addition of oxygen in a benzo[b]oxazine structure (Compound 14, 4.6μM) retained activity; and like the piperidine scaffold, deletion of thephenyl portion led to an inactive compound (morpholine, Compound 15,inactive). Addition of a phenyl substitution did not bring backactivity, unlike in the piperidine scaffold, Compound 16. Finally,moving the nitrogen outside of the ring system led to an inactivecompound (Compound 17) and other smaller ring systems were nottolerated, Compound 18. Compound 18, VU937, inhibited the AnKir1 channelwith activity 60-fold less than VU041 with an IC₅₀ of 29,670 nM (95% CI:17,680-49,770 nM).

TABLE 1 Selectivity of VU041 against mosquito and mammalian Kirchannels. IC₅₀ values were determined by thallium-flux assay, and areexpressed as means (n = 3). Selectivity ratios (SR) are expressed as:AnKir1 IC50/secondary Kir IC₅₀. Mean IC₅₀ values were compared by aone-way ANOVA followed by Tukey's posttest using InStat ™ (GraphPadSoftware, San Diego CA, USA). Kir Channel IC₅₀ μM (95% Cl) SelectivityRatio AnKir1  2.5 (1.1-3.6) ^(A) — AeKir1  1.7 (0.6-2.8) ^(A) 0.7Kir1.1 >30 ^(B) >12 Kir2.1 12.7 (10.2-14.3) ^(C) 5 Kir4.1 >30 ^(B) >12Kir6.2 + SUR1 >30 ^(B) >12 Kir7.1 >30 ^(B) >12 Capital lettersuperscripts (A, B, C) indicate statistical categorization of the means

TABLE 2 Structure-activity relationship (SAR) for the “left- hand”portion of VU041.

AnKir1 hKir2.1 IC₅₀ IC₅₀ Cmpd VU# R (μM) (μM) 1 VU0048041

2.3 11.3 2 VU0650728

Inactive N.D. 3 VU0650727

inactive N.D. 4 VU0650729

12.1 >30 5 VU0650730

2.4 >30 6 VU0657111

8.0 N.D. 7 VU0657112

Inactive N.D.

TABLE 3 Structure-activity relationship (SAR) for the “right-hand”portion of VU041.

AnKir1 hKir2.1 IC₅₀ IC₅₀ Cmpd VU# R (μM) (μM) 1 VU0048041

2.3 N.D. 8 VU0652933

15 N.D. 9 VU0652954

6.7 >30 10 VU0652944

Inactive N.D. 11 VU0652947

Inactive N.D. 12 VU0652942

5.6 >30 13 VU0652943

4.5 >30 14 VU0652934

4.6 N.D. 15 VU0299695

Inactive N.D. 16 VU0652955

Inactive N.D. 17 VU0652931

Inactive N.D. 18 VU0652937

inactive N.D.

TABLE 4 Chemical composition of solutions used in Xenopus oocyteexperiments. Solution # I II III NaCl 96 88.5 88.5 NMDG-Cl 0 9.5 0 KCl 20.5 10 MgCl2 1.0 1.0 1.0 CaCl2 1.8 1.8 1.8 HEPES 5 5 5 The pH of allsolutions was adjusted to 7.5 with NMDG-OH. The osmolality of eachsolution was verified to be 190 ± 5 mOsm/kg H2O by vapor pressureosmometry. NMDG = N-methyl D-glucamine.

TABLE 5 Mean (n = 5) ED₅₀ (μg/mg of mosquito) after topical exposure toVU041 with and without the synergists Piperonyl butoxide andS,S,S-tributyl phosphorotrithioate (500 ng/insect) in adult female An.gambiae. Mosquitoes were pre-treated with PBO and DEF four hours priorto VU041 treatment. Mosquito strains not labeled by the same letter(e.g., A or B) indicates statistical significance (P < 0.05) of themeans as determined by a one-way ANOVA with Tukey's post-hoc test.²Resistance Compound(s) ¹G3 Strain ¹Akron Strain Ratio VU041  1.8(0.9-3.1) A  2.8 (2.1-4.1) A 1.5 VU041 + PBO 0.88 (0.3-1.4) B 0.37(0.2-0.7) B 0.4 VU041 + DEF  1.7 (1.1-2.2) A N.D. N.D. VU730  2.4(2.1-2.9) A N.D. N.D. VU730 + PBO  0.9 (0.4-1.5) B N.D. N.D.VU937 >10 >10 N.D. Permethrin* 0.03e−5 1.0e−3 33 (0.02e−0.05e−5)(0.8e−4-1.3e−3) ¹ED₅₀, μg/mg (95% confidence limits). ²RR: Resistanceratio for G3 = ED₅₀ Akron/ED₅₀ G3 N.D. = no data *Topical ED₅₀ valuesfrom reference ²¹.

High Throughput Screening.

To perform Tl+ flux assays, stably transfected T-Rex-HEK-293 cellsexpressing AnKir1 channels were cultured overnight in 384-well plates(20,000 cells/20 μL/well), black-walled, clear-bottomed BD Pure Coatamine-coated plates (BD, Bedford, Mass.) with a plating media containingDMEM, 10% dialyzed FBS and 1 μg/mL tetracycline. Approximatelytwenty-four hours after cell plating, the cell culture medium wasreplaced with a dye-loading solution containing assay buffer (HanksBalanced Salt Solution with 20 mM HEPES, pH 7.3), 0.01% (w/v) PluronicF-127 (Life Technologies, Carlsbad, Calif.), and 1.2 μM of thethallium-sensitive dye Thallos-AM (TEFlabs, Austin, Tex.). Following 1hr incubation at room temperature, the dye-loading solution was washedfrom the plates and replaced with 20 μL/well of assay buffer.

Whole-Cell Patch Clamp Electrophysiology.

Transiently transfected HEK-293T cells expressing AnKir1 werevoltage-clamped in the whole-cell configuration of the patch clamptechnique. The extracellular bath solution contained (in mM): 135 NaCl,5 KCl, 2 CaCl₂), 1 MgCl₂, 5 glucose, 10 HEPES free acid, pH 7.4, 290mOsm/kg H₂O. The pipette solution contained (in mM): 135 KCl, 2 MgCl₂, 1EGTA, 10 HEPES free acid, 2 Na₂ATP (Roche, Indianapolis, Ind.), pH 7.3,275 mOsm. Cells were voltage clamped at −75 mV, stepped to −120 mV for200 msec, and then ramped to 120 mV at a rate of 2.4 mV/msec.Concentration-response curves (CRCs) were constructed by measuring theeffects of increasing doses of inhibitors on AnKir1 currents at −120 mV.All recordings were made at room temperature (20-23° C.).

Two-Electrode Voltage Clamp Electrophysiology.

Heterologous expression of AeKir1 or AeKir2B was performed in Xenopuslaevis oocytes. Current-voltage (1-V) relationships of oocytes weremeasured by clamping the membrane potential (V_(m)) near thespontaneous, resting potential and then initiating a voltage-steppingprotocol (via the Clampex module of pCLAMP) with 20 mV steps from −140mV to +40 mV (100 ms each).

Mosquito Colonies.

An. gambiae mosquitoes, G3 strain (MRA-112), were reared in anenvironmental chamber at 27° C. and 75% relative humidity at VanderbiltUniversity, Department of Biological Sciences, Nashville, Tenn. An.gambiae, Akron strain (MRA-913, isolated in Benin), was reared in aseparate environmental chamber at the Emerging Pathogens Institute,University of Florida, Gainesville, Fla. at 27° C. and 75% relativehumidity. The Akron strain of An. gambiae was selected every 5thgeneration for anticholinergic and pyrethroid resistance by exposingadult mosquitoes to bendiocarb (12.5 μg/bottle) and permethrin (21.5μg/bottle) using the CDC bottle assay. Survivors of both sexes were thenmixed and allowed to breed. To ensure resistance, the mosquitoes fromeach resistant egg cohort were exposed to an LC99 dose (based on G3toxicity values) of permethrin and propoxur. The mosquitoes used in thisstudy were derived from the same colony that had previously been used todemonstrate the resistance of the Akron strain to propoxur andpermethrin, resistance that has been attributed to both target site andmetabolic resistance through upregulation of CYP450 enzymes.

An established colony of Ae. aegypti mosquitoes, Liverpool strain(LVP-1612, MRA-735), was reared and maintained in an environmentalchamber at 28° C. and 80% relative humidity at the Ohio AgriculturalResearch and Development Center (OARDC) of The Ohio State University,Wooster, Ohio. When needed, eggs from a pyrethroid-resistant strain ofAe. aegypti, Puerto Rico strain (PR, NR-48830), were obtained from BEIResources, NIAID, NIH and reared to adulthood. Third-instar larvae ofthe resistant strain of Ae. aegypti were exposed to permethrin (0.1mg/ml) every third generation to maintain the resistance trait. Adultmosquitoes of all strains were fed a 10% sucrose solution ad libitum andheld under a 12h/12h light cycle. All experiments were carried out onadult females 3-5 days post-emergence.

Toxicology Experiments in An. gambiae.

Topical toxicity bioassays were performed on non-blood-fed adult femalemosquitoes. The mosquitoes were chilled on ice for 1-3 minutes, duringwhich 200 nL of compound (dissolved in 95% ethanol) was applied onto theabdomen of each insect with a handheld Hamilton® micro-applicator. Forsynergism studies, 500 ng of the potential synergist (e.g., piperonylbutoxide [PBO] or S,S,S-tributyl phosphorotrithioate [DEF]) permilligram of mosquito (ng/mg) was applied to the abdomen 4 h prior toapplication of the insecticide. For each compound, 6-8 doses thatresulted in toxicity ranging between 0% and 100% were applied to aminimum of 30 mosquitoes each, repeated 3 times on different mosquitobroods. The three resulting ED₅₀ values were averaged. An ethanol-onlytreatment was included in each experiment as a negative control. Treatedmosquitoes were transferred to small cages with access to 10% sucroseand held under rearing conditions for 24 h. Mortality was recorded at 24h. Mortality data were pooled and analyzed by log-probit using Poloplus®to determine 24 h ED₅₀ values, after correcting for control mortalityusing Abbot's formula.

Blood meal processing studies were performed with similar methods, withthe exception that An. gambiae were blood-fed on anesthetized mice. Allmethods were carried out in accordance with Vanderbilt InstitutionalAnimal Care and Use Committee approval. Upon completion of bloodfeeding, female mosquitoes with fully distended abdomens were selected,and 200 nL of compound at a non-lethal concentration (1 μg/mg) wasapplied directly to the abdomen. Any mosquito that died during the 24 hobservation period was excluded from the analysis. Images of theabdomens were acquired at 0, 2, 5, 8, and 24 h through the dorsalcuticle; abdomens were measured at the widest point. Images werecaptured using bright-field illumination on a Nikon 90i light microscope(Nikon Corp., Tokyo, Japan) connected to a Photometrics CoolSNAP HQ2high-sensitivity monochrome CCD camera (Roper Scientific, Ottobrunn,Germany). Digital images were acquired using Nikon Advanced ResearchNIS-Elements software. Mean abdominal diameters were compared usingone-way ANOVA with a Tukey's post-hoc analysis (Prism 6, GraphpadSoftware, La Jolla, Calif.).

Toxicity Experiments in Ae. aegypti.

Topical toxicity bioassays in adult female Ae. aegypti (LVP and PRstrains) were performed. For a given dose, 10 non-blood-fed mosquitoeswere immobilized on ice, and 500 nL of VU041 was applied to the thoraxof each using a handheld Hamilton® microapplicator. A solvent-onlytreatment was included in each experiment as a negative control. Treatedmosquitoes were transferred to small cages with access to 10% sucroseand held under rearing conditions for 24 h. The efficacy of a dose wasmeasured as the percentage of treated mosquitoes in a cage that wereflightless or dead at 24 h. Four to eight replicates of 10 mosquitoeswere performed per dose. The ED₅₀ values were determined using anon-linear curve fit analysis (log [inhibitor] vs. responsevariable-slope) in Prism 6 (Graphpad Software).

The efficacy of VU041 was compared to that of its inactive analog VU937in LVP mosquitoes (only). In these experiments, two groups of 10mosquitoes were treated with a dose of VU041 or VU937 at the approximateED₅₀ of VU041 (3.24 μg/mg mosquito), and the mosquitoes' conditions wereassessed 24 h later. Six replicate experiments of 10 mosquitoes eachwere performed. The mean efficacies of the solvent, VU041, and VU937were analyzed using a one-way ANOVA with a Newman-Keuls post hocanalysis (Prism 6, Graphpad Software).

Diuresis Experiments in Ae. aegypti.

The excretory capacity of adult female Ae. aegypti (LVP strain) wasmeasured in groups of 5 mosquitoes treated with a sub-lethal dose ofVU041 (1.7 μg/mg mosquito), VU937 (1.7 μg/mg mosquito), or solvent 2 hbefore injecting the hemolymph of each mosquito with 900 nL of apotassium-enriched, phosphate-buffered saline (K⁺-PBS) using a NanojectII microinjector (Drummond Scientific Company, Broomall, Pa.). TheK⁺-PBS contained the following (in mM): 92.2 NaCl, 47.5 KCl, 10 Na₂HPO₄,and 2 KH₂PO₄ (pH 7.5). Each treatment group of 5 mosquitoes wastransferred into a separate graduated, packed-cell volume tube (MidSci,St. Louis, Mo.) and held for 1 h at 28° C. The volume excreted by themosquitoes was measured visually via the graduated column at the bottomof the tube. At least 8 replicates (5 mosquitoes per replicate) wereperformed for each treatment. All mosquitoes were confirmed to be aliveat the end of 1h. The mean volumes excreted by solvent-, VU041-, andVU937-treated mosquitoes were analyzed using one-way ANOVA with aNewman-Keuls post hoc analysis (Prism 6, Graphpad Software).

Mosquito Fecundity Experiments.

The effects of VU041 on fecundity in An. gambiae were determined.Briefly, adult female mosquitoes were given access to an anesthetizedmouse for 60 min. After 60 min., engorged mosquitoes were immobilized onice; and 200 nL of VU041 (ED₃₀: 1 μg/mg of mosquito), VU937 (10 μg/mg ofmosquito), or solvent was applied directly to the abdomen. Aftertreatment, individual female mosquitoes were transferred to Drosophilavials (Fisher Scientific, Pittsburgh, OA) containing 2 mL of water. Thetotal number of eggs was counted 72 hours after each mosquito wastransferred into a vial. Any mosquitoes that died during this 72-hourperiod were excluded from the analysis. All assays were performed in anenvironmental chamber that was maintained at 27° C. and 75% relativehumidity; mosquitoes were given access to 10% sucrose solution adlibitum. At least 25 female mosquitoes were used per replicate for eachtreatment group; each treatment was repeated on three separate broods,giving a total number of individuals from 75-113 for each group.

To determine the effects of VU041 on fecundity in Ae. aegypti, adultfemale mosquitoes were allowed to feed for 1h on heparinized rabbitblood (Hemostat) presented in a membrane feeder (Hemotek). After thefeeding period, the mosquitoes were immobilized on ice, visuallyinspected for blood engorgement, and topically treated with 500 nL ofsolvent, VU041 (3.4 μg/mg mosquito), or VU937 (3.4 μg/mg mosquito). Themosquitoes were returned to rearing conditions for 24 h, after whichthey were transferred to individual egg-laying glass tubes 21 mm×70 mm(Fisher Scientific, Pittsburgh, Pa.) with a piece of coffee filter(Melitta USA, Clearwater, Fla.) cut to fit the bottom of the tube. Thefilter was wetted with 150 μl of dH₂O, and the open end of the tube wasplugged with a cotton ball. The mosquitoes in their individual egglaying tubes were returned to rearing conditions for an additional 48 h,and the number of eggs laid was counted. Any mosquitoes that died duringthe 72 h period after blood feeding were excluded from the analysis.Thirty female mosquitoes were used per replicate for each treatmentgroup; each treatment was repeated on four separate broods, giving atotal number of individuals from 87-113 for each treatment group. Forboth species, the median number of eggs laid per mosquito was comparedusing a Kruskal-Wallis ANOVA with a Dunn's post-hoc analysis (Prism 6,Graphpad Software).

Honeybee Rearing and Toxicity Experiments.

Frames of late-stage honeybee (A. mellifera) pupae were taken from fourcolonies at The Ohio State University Honey Bee Lab in Wooster, Ohio,and maintained in a dark humid incubator at 34° C. (Darwin Chambers Co.,St. Louis, Mo., model H024) until adult bees emerged. New adults werebrushed from frames daily, placed in wooden screen cages (21×14×12 cm),and provided with 1:1 (w/w) sucrose in water.

Acute toxicity experiments in adult bees were performed as follows:Twenty-four hours after emergence, adult honey bees in cages wereanaesthetized with carbon dioxide, divided into groups of approximately20 bees each, and placed in plastic-coated paper cups (177 cm³; UNIQPaper Yogurt Cup, Frozen Dessert Supplies, Gilbert, Ariz.) covered withcotton cheesecloth. Smaller groups of bees were anaesthetized a secondtime and dosed on the thoracic notum with 10 μl of VU041 (100 μg/μl) or10 μl of vehicle. As a positive control, some bees were treated with 3μl of bifenthrin (0.1 μg/μl). Negative control bees were treated with 3μl of solvent. Applications were made using a 20 μl micropipette(Fisherbrand Finnpipette F2, Fisher Scientific, Pittsburgh, Pa.) with adisposable plastic tip. After treatment, bees were returned to the papercups and provided with sugar syrup in punctured 1.5 ml microcentrifugetubes. Toxicity to honey bees was recorded at 48 h, rather than the 24 hused for mosquitoes, to allow for the possibility of delayed toxicitydue to the larger body size of the bee. Bees showing no movement 48 hafter treatment were scored as dead. A Fisher's Exact Test was used tocompare the proportion of mortality induced by VU041 or bifenthrincompared to their respective vehicle controls.

Structure-Activity Relationships of Pinacidil Analogs to Salivary GlandFunction in Amblyomma americanum.

We modified the structure of pinacidil to form analogs, and tested theeffect of the analogs on salivary function in the tick Amblyommaamericanum. Results are shown in Table 6.

TABLE 6 Effect on Salivation Name Ar X R Class at 10 minutes Pinacidil4-pyridyl NCN (±)CHMeCMe₃ cyanoguanidine Reduced ~98% DRS-1 4-pyridyl S(S)-CHMeCMe₃ thiourea Reduced ~80% DRS-2 4-pyridyl O CH₂CMe₃ urea Nochange DRS-3 3-pyridyl NCN (±)CHMeCMe₃ cyanoguanidine Reduced ~100%DRS-4 4-pyridyl E-CHNO₂ CH₂CMe₃ nitroethenediannine Reduced ~83%

Insects and Arachnids that May be Targeted by this Invention.

This invention may be used against any blood-feeding or sap-feedinginsects or arachnids, including (in each case, where applicable—not allmembers of a taxon necessarily feed on blood or sap): members of theorder Diptera, for example Aedes spp., Agromyza spp., Anastrepha spp.,Anopheles spp., Asphondylia spp., Bactrocera spp., Bibio hortulanus,Calliphora erythrocephala, Calliphora vicina, Ceratitis capitata,Chironomus spp., Chrysomyia spp., Chrysops spp., Chrysozona pluvialis,Cochliomyia spp., Contarinia spp., Cordylobia anthropophaga, Cricotopussylvestris, Culex spp., Culicoides spp., Culiseta spp., Cuterebra spp.,Dacus oleae, Dasyneura spp., Delia spp., Dermatobia hominis, Drosophilaspp., Echinocnemus spp., Fannia spp., Gasterophilus spp., Glossina spp.,Haematopota spp., Hydrellia spp., Hydrellia griseola, Hylemya spp.,Hippobosca spp., Hypoderma spp., Liriomyza spp., Lucilia spp., Lutzomyiaspp., Mansonia spp., Musca spp., Oestrus spp., Oscinella frit,Paratanytarsus spp., Paralauterborniella subcincta, Pegomyia spp.,Phlebotomus spp., Phorbia spp., Phormia spp., Piophila casei,Prodiplosis spp., Psila rosae, Rhagoletis spp., Sarcophaga spp.,Simulium spp., Stomoxys spp., Tabanus spp., Tetanops spp., and Tipulaspp.; and members of the order Hemiptera, for example Acyrthosiphononobrychis, Acyrthosiphon pisum, Adelges laricis, Aonidiella aurantii,Aphidula nasturtii, Aphis fabae, Aphis gossypii, Aphis pomi, Aulacorthumsolani, Bemisia tabaci, Brachycaudus cardui, Brevicoryne brassicae,Dalbulus maidis, Dreyfusia nordmannianae, Dreyfusia piceae, Dysaphisradicola, Empoasca fabae, Eriosoma lanigerum, Laodelphax striatella,Macrosiphum avenae, Macrosiphum euphorbiae, Macrosiphon rosae, Megouraviciae, Metopolophium dirhodum, Myzus persicae, Myzus cerasi,Nephotettix cincticeps, Nilaparvata lugens, Perkinsiella saccharicida,Phorodon humuli, Psylla mali, Psylla pini, Psylla pyricola,Rhopalosiphum maidis, Schizaphis graminum, Sitobion avenae, Sogatellafuncifera, Toxoptera citricida, Trialeurodes abutilonea, Trialeurodesvaporariorum, and Viteus vitifolii; Coleoptera, for example Anthonomusgrandis; and arachnids, for example Acarus spp., Aceria sheldoni,Aculops spp., Aculus spp., Amblyomma spp., Amphitetranychus viennensis,Argas spp., Boophilus spp., Brevipalpus spp., Bryobia graminum, Bryobiapraetiosa, Centruroides spp., Chorioptes spp., Dermanyssus gallinae,Dermatophagoides pteronyssinus, Dermatophagoides farinae, Dermacentorspp., Eotetranychus spp., Epitrimerus pyri, Eutetranychus spp.,Eriophyes spp., Glycyphagus domesticus, Halotydeus destructor,Hemitarsonemus spp., Hyalomma spp., Ixodes spp., Latrodectus spp.,Loxosceles spp., Metatetranychus spp., Neutrombicula autumnalis,Nuphersa spp., Oligonychus spp., Ornithodorus spp., Ornithonyssus spp.,Panonychus spp., Phyllocoptruta oleivora, Polyphagotarsonemus latus,Psoroptes spp., Rhipicephalus spp., Rhizoglyphus spp., Sarcoptes spp.,Scorpio maurus, Steneotarsonemus spp., Steneotarsonemus spinki,Tarsonemus spp., Tetranychus spp., Trombicula alfreddugesi, Vaejovisspp., and Vasates lycopersici.

Compositions Containing Compounds of the Invention.

The compounds of the invention are suitable for use on any plant,including but not limited to those that have been genetically modifiedto be resistant to active ingredients such as herbicides, or to producebiologically active compounds that control infestation by plant pests,e.g. “BT cotton.”

A compound of the invention may be used in mixtures with fertilizers(for example nitrogen-, potassium- or phosphorus-containingfertilizers). Suitable formulation types include granules of fertilizer.The mixtures preferably contain up to 25% by weight of the compound ofthe invention. The invention therefore also provides a fertilizercomposition comprising a fertilizer and a compound of the invention.

The compositions of this invention may contain other compounds havingbiological activity, for example micronutrients or compounds havingfungicidal activity or which possess plant growth regulating,herbicidal, insecticidal, nematicidal or acaricidal activity.

The compound of the invention may be the sole active ingredient of acomposition or it may optionally be admixed with one or more additionalactive ingredients such as another pesticide, e.g. another insecticide,fungicide, or herbicide, or a synergist or plant growth regulator whereappropriate. An additional active ingredient may provide a compositionhaving a broader spectrum of activity or increased persistence at alocus; it may synergize the activity or complement the activity (forexample by increasing the speed of effect or overcoming repellency) of acompound; or help to overcome or prevent the development of resistanceto individual components. The particular additional active ingredientwill depend upon the intended utility of the composition. Examples ofsuitable additional pesticide compounds include the following: a)Pyrethroids, such as permethrin, cypermethrin, fenvalerate,esfenvalerate, deltamethrin, cyhalothrin (in particularlambda-cyhalothrin and gamma cyhalothrin), bifenthrin, fenpropathrin,cyfluthrin, tefluthrin, fish safe pyrethroids (for example ethofenprox),natural pyrethrin, tetramethrin, S-bioallethrin, fenfluthrin,prallethrin, acrinathirin, etofenprox or5-benzyl-3-furylmethyl-(E)-(1R,3S)-2,2-dimethyl-3-(2-oxothiolan-3-ylidene-methyl)cyclopropanecarboxylate; b) Organophosphates, such as profenofos, sulprofos,acephate, methyl parathion, azinphos-methyl, demeton-s-methyl,heptenophos, thiometon, fenamiphos, monocrotophos, profenofos,triazophos, methamidophos, dimethoate, phosphamidon, malathion,chlorpyrifos, phosalone, terbufos, fensulfothion, fonofos, phorate,phoxim, pirimiphos-methyl, pirimiphos-ethyl, fenitrothion, fosthiazateor diazinon; c) Carbamates (including aryl carbamates), such aspirimicarb, triazamate, cloethocarb, carbofuran, furathiocarb,ethiofencarb, aldicarb, thiofurox, carbosulfan, bendiocarb, fenobucarb,propoxur, methomyl or oxamyl; d) Benzoyl ureas, such as diflubenzuron,triflumuron, hexaflumuron, flufenoxuron, diafenthiuron, lufeneron,novaluron, noviflumuron or chlorfluazuron; e) Organo-tin compounds, suchas cyhexatin, fenbutatin oxide or azocyclotin; f) Pyrazoles, such astebufenpyrad, tolfenpyrad, ethiprole, pyriprole, fipronil, andfenpyroximate; g) Macrolides, such as avermectins or milbemycins, forexample abamectin, emamectin benzoate, ivermectin, milbemycin, spinosad,azadirachtin, milbemectin, lepimectin or spinetoram; h) Hormones orpheromones; i) Organochlorine compounds, such as endosulfan (inparticular alpha-endosulfan), benzene hexachloride, DDT, chlordane ordieldrin; j) Amidines, such as chlordimeform or amitraz; k) Fumigantagents, such as chloropicrin, dichloropropane, methyl bromide or metam,l) Neonicotinoid compounds, such as imidacloprid, thiacloprid,acetamiprid, nitenpyram, dinotefuran, thiamethoxam, clothianidin, ornithiazine; m) Diacylhydrazines, such as tebufenozide, chromafenozide ormethoxyfenozide; n) Diphenyl ethers, such as diofenolan or pyriproxifen;o) Ureas such as Indoxacarb or metaflumizone; p) Ketoenols, such asSpirotetramat, spirodiclofen or spiromesifen; q) Diamides, such asflubendiamide, chlorantraniliprole (Rynaxypyr®) or cyantraniliprole; r)Essential oils such as Bugoil®—(Plantlmpact); or s) a compound selectedfrom buprofezine, flonicamid, acequinocyl, bifenazate, cyenopyrafen,cyflumetofen, etoxazole, flometoquin, fluacrypyrim, fluensulfone,flufenerim, flupyradifuone, harpin, iodomethane, dodecadienol,pyridaben, pyridalyl, pyrimidifen, flupyradifurone,4-[(6-Chloro-pyridin-3-ylmethyl)-(2,2-difluoro-ethyl)-amino]-5H-furan-2-one(DE 102006015467), CAS: 915972-17-7 (WO 2006129714; WO2011/147953;WO2011/147952), CAS: 26914-55-8 (WO 2007020986), chlorfenapyr,pymetrozine, sulfoxaflor and pyrifluqinazon.

In addition to the major chemical classes of pesticide listed above,other pesticides having particular targets may optionally be employed inthe composition, if appropriate for the intended use of the composition.For instance, selective insecticides for particular crops, for examplestem borer specific insecticides (such as cartap) or hopper specificinsecticides (such as buprofezin) for use in rice may be employed.Alternatively, insecticides or acaricides specific for particular insectspecies/stages may also be included in the compositions (for exampleacaricidal ovo-larvicides, such as clofentezine, flubenzimine,hexythiazox or tetradifon; acaricidal motilicides, such as dicofol orpropargite; acaricides, such as bromopropylate or chlorobenzilate; orgrowth regulators, such as hydramethylnon, cyromazine, methoprene,chlorfluazuron or diflubenzuron).

Examples of fungicidal compounds which may optionally be included in thecomposition of the invention are(E)-N-methyl-2-[2-(2,5-dimethylphenoxymethyl)phenyl]-2-methoxy-iminoacetamide(SSF-129),4-bromo-2-cyano-N,N-dimethyl-6-trifluoromethylbenzimidazole-1-sulfonamide-,.alpha.-[N-(3-chloro-2,6-xylyl)-2-methoxyacetamido]-.gamma.-butyrolacton-e,4-chloro-2-cyano-N,N-dimethyl-5-p-tolylimidazole-1-sulfonamide (IKF-916,cyamidazosulfamid),3-5-dichloro-N-(3-chloro-1-ethyl-1-methyl-2-oxopropyl)-4-methylbenzamide(RH-7281, zoxamide),N-allyl-4,5,-dimethyl-2-trimethylsilylthiophene-3-carboxamide(MON65500),N-(1-cyano-1,2-dimethylpropyl)-2-(2,4-dichlorophenoxy)propionamide(AC382042), N-(2-methoxy-5-pyridyl)-cyclopropane carboxamide,acibenzolar (CGA245704) (e.g. acibenzolar-S-methyl), alanycarb,aldimorph, anilazine, azaconazole, azoxystrobin, benalaxyl, benomyl,benthiavalicarb, biloxazol, bitertanol, bixafen, blasticidin S,boscalid, bromuconazole, bupirimate, captafol, captan, carbendazim,carbendazim chlorhydrate, carboxin, carpropamid, carvone, CGA41396,CGA41397, chinomethionate, chlorothalonil, chlorozolinate, clozylacon,copper containing compounds such as copper oxychloride, copperoxyquinolate, copper sulfate, copper tallate and Bordeaux mixture,cyclufenamid, cymoxanil, cyproconazole, cyprodinil, debacarb,di-2-pyridyl disulfide 1,1′-dioxide, dichlofluanid, diclomezine,dicloran, diethofencarb, difenoconazole, difenzoquat, diflumetorim,O,O-di-iso-propyl-S-benzyl thiophosphate, dimefluazole, dimetconazole,dimethomorph, dimethirimol, diniconazole, dinocap, dithianon, dodecyldimethyl ammonium chloride, dodemorph, dodine, doguadine, edifenphos,epoxiconazole, ethirimol, ethyl-({umlaut over(Z)})-N-benzyl-N-([methyl(methyl-thioethylideneamino-oxycarbonyl)amino]thio)-.beta.-alaninate,etridiazole, famoxadone, fenamidone (RPA407213), fenarimol,fenbuconazole, fenfuram, fenhexamid (KBR2738), fenpiclonil, fenpropidin,fenpropimorph, fentin acetate, fentin hydroxide, ferbam, ferimzone,fluazinam, fludioxonil, flumetover, fluopyram, fluoxastrobin,fluoroimide, fluquinconazole, flusilazole, flutolanil, flutriafol,fluxapyroxad, folpet, fuberidazole, furalaxyl, furametpyr, guazatine,hexaconazole, hydroxyisoxazole, hymexazole, imazalil, imibenconazole,iminoctadine, iminoctadine triacetate, ipconazole, iprobenfos,iprodione, iprovalicarb (SZX0722), isopropanyl butyl carbamate,isoprothiolane, isopyrazam, kasugamycin, kresoxim-methyl, LY186054,LY211795, LY248908, mancozeb, mandipropamid, maneb, mefenoxam,metalaxyl, mepanipyrim, mepronil, metalaxyl, metconazole, metiram,metiram-zinc, metominostrobin, myclobutanil, neoasozin, nickeldimethyldithiocarbamate, nitrothal-isopropyl, nuarimol, ofurace,organomercury compounds, oxadixyl, oxasulfuron, oxolinic acid,oxpoconazole, oxycarboxin, pefurazoate, penconazole, pencycuron,penflufen, penthiopyrad, phenazin oxide, phosetyl-Al, phosphorus acids,phthalide, picoxystrobin (ZA1963), polyoxinD, polyram, probenazole,prochloraz, procymidone, propamocarb, propiconazole, propineb, propionicacid, prothioconazole, pyrazophos, pyrifenox, pyrimethanil,pyraclostrobin, pyroquilon, pyroxyfur, pyrrolnitrin, quaternary ammoniumcompounds, quinomethionate, quinoxyfen, quintozene, sedaxane,sipconazole (F-155), sodium pentachlorophenate, spiroxamine,streptomycin, sulfur, tebuconazole, tecloftalam, tecnazene,tetraconazole, thiabendazole, thifluzamid,2-(thiocyanomethylthio)benzothiazole, thiophanate-methyl, thiram,timibenconazole, tolclofos-methyl, tolylfluanid, triadimefon,triadimenol, triazbutil, triazoxide, tricyclazole, tridemorph,trifloxystrobin (CGA279202), triforine, triflumizole, triticonazole,validamycin A, vapam, vinclozolin, zineb and ziram,N-[9-(dichloromethylene)-1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-yl]-3--(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide[1072957-71-1],1-methyl-3-difluoromethyl-1H-pyrazole-4-carboxylic acid(2-dichloromethylene-3-ethyl-1-methyl-indan-4-yl)-amide, and1-methyl-3-difluoromethyl-4H-pyrazole-4-carboxylic acid[2-(2,4-dichloro-phenyl)-2-methoxy-1-methyl-ethyl]-amide.

In addition, biological agents may be included in the composition of theinvention e.g. Bacillus species such as Bacillus firmus, Bacilluscereus, Bacillus subtilis, and Pasteuria species such as Pasteuriapenetrans and Pasteuria nishizawae. A suitable Bacillus firmus strain isstrain CNCM 1-1582 which is commercially available as BioNem™. Asuitable Bacillus cereus strain is strain CNCM 1-1562. For both Bacillusstrains more details can be found in U.S. Pat. No. 6,406,690. Otherbiological organisms that may be included in the compositions of theinvention are bacteria such as Streptomyces spp. such as S. avermitilis,and fungi such as Pochonia spp. such as P. chlamydosporia. Also ofinterest are Metarhizium spp. such as M. anisopliae; Pochonia spp. suchas P. chlamydosporia.

The compounds of the invention may be mixed with soil, peat or otherrooting media for the protection of plants against seed-borne,soil-borne or foliar fungal diseases.

Examples of suitable synergists for use in the compositions includepiperonyl butoxide, sesamex, safroxan and dodecyl imidazole.

Suitable herbicides and plant-growth regulators for inclusion in thecompositions will depend upon the intended target and the effectrequired.

An example of a rice selective herbicide which may be included ispropanil. An example of a plant growth regulator for use in cotton isPIX™.

Some mixtures may comprise active ingredients which have significantlydifferent physical, chemical or biological properties such that they donot easily lend themselves to the same conventional formulation type. Inthese circumstances other formulation types may be prepared. Forexample, where one active ingredient is a water insoluble solid and theother a water insoluble liquid, it may nevertheless be possible todisperse each active ingredient in the same continuous aqueous phase bydispersing the solid active ingredient as a suspension but dispersingthe liquid active ingredient as an emulsion. The resultant compositionis a suspoemulsion (SE) formulation.

Unless otherwise stated the weight ratio of compounds of the inventionwith an additional active ingredient may generally be between 1000:1 and1:1000. In other embodiments that weight ratio may be between 500:1 to1:500, for example between 100:1 to 1:100, for example between 1:50 to50:1, for example 1:20 to 20:1, for example 1:10 to 10:1, for example1:5 to 5:1, for example 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, or 5:1.

Compositions of the invention include those prepared by premixing priorto application, e.g. as a ready mix or tank mix, or by simultaneousapplication or sequential application to the plant.

In order to apply a compound of the invention as an insecticide oracaricide to a pest, a locus of pest, or to a plant susceptible toattack by a pest, a compound of the invention may be formulated into acomposition which includes, in addition to the active compound, asuitable inert diluent or carrier and, optionally, a surface activeagent or surfactant (SFA). SFAs are compounds that can modify theproperties of an interface (for example, a liquid/solid, liquid/air, orliquid/liquid interface) by lowering the interfacial tension, therebyleading to changes in other properties (for example dispersion,emulsification and wetting). It is preferred that such compositions(both solid and liquid formulations) comprise, by weight, 0.0001 to 95%,more preferably 1 to 85%, for example 5 to 60%, of that of a compound ofthe invention. The composition may be used for the control of pests suchthat a compound of the invention is applied at a rate of from 0.1 g to10 kg per hectare, preferably from 1 g to 6 kg per hectare, morepreferably from 1 g to 1 kg per hectare.

In one embodiment the compounds of the invention are used for pestcontrol on cotton or other crop plants at 1:500 g/ha, for example 10-70g/ha.

When used in a seed dressing, a compound of the invention is used at arate of 0.0001 g to 10 g (for example 0.001 g or 0.05 g), preferably0.005 g to 10 g, more preferably 0.005 g to 4 g, per kilogram of seed.

Compositions comprising a compound of the invention can be chosen from anumber of formulation types known in the art, including dustable powders(DP), soluble powders (SP), water soluble granules (SG), waterdispersible granules (WG), wettable powders (WP), granules (GR) (slow orfast release), soluble concentrates (SL), oil miscible liquids (OL),ultra-low volume liquids (UL), emulsifiable concentrates (EC),dispersible concentrates (DC), emulsions (both oil in water (EW) andwater in oil (EO)), micro-emulsions (ME), suspension concentrates (SC),aerosols, fogging/smoke formulations, capsule suspensions (CS) and seedtreatment formulations. The formulation type chosen in any instance willdepend upon the particular purpose and the physical, chemical andbiological properties of the compound of the invention.

Dustable powders (DP) may be prepared by mixing a compound of theinvention with one or more solid diluents (for example natural clays,kaolin, pyrophyllite, bentonite, alumina, montmorillonite, kieselguhr,chalk, diatomaceous earths, calcium phosphates, calcium and magnesiumcarbonates, sulfur, lime, flours, talc and other organic and inorganicsolid carriers) and mechanically grinding the mixture to a fine powder.

Soluble powders (SP) may be prepared by mixing a compound of theinvention with one or more water-soluble inorganic salts (such as sodiumbicarbonate, sodium carbonate or magnesium sulfate) or one or morewater-soluble organic solids (such as a polysaccharide) and, optionally,one or more wetting agents, one or more dispersing agents or a mixtureof said agents to improve water dispersibility/solubility. The mixtureis then ground to a fine powder Similar compositions may also begranulated to form water soluble granules (SG).

Wettable powders (WP) may be prepared by mixing a compound of theinvention with one or more solid diluents or carriers, one or morewetting agents and, preferably, one or more dispersing agents and,optionally, one or more suspending agents to facilitate the dispersionin liquids. The mixture is then ground to a fine powder. Similarcompositions may also be granulated to form water dispersible granules(WG).

Granules (GR) may be formed either by granulating a mixture of acompound of the invention and one or more powdered solid diluents orcarriers, or from pre-formed blank granules by absorbing a compound ofthe invention (or a solution thereof, in a suitable agent) in a porousgranular material (such as pumice, attapulgite clays, fuller's earth,kieselguhr, diatomaceous earths or ground corn cobs) or by adsorbing acompound of the invention (or a solution thereof, in a suitable agent)on to a hard core material (such as sands, silicates, mineralcarbonates, sulfates or phosphates) and drying if necessary. Agentswhich are commonly used to aid absorption or adsorption include solvents(such as aliphatic and aromatic petroleum solvents, alcohols, ethers,ketones and esters) and sticking agents (such as polyvinyl acetates,polyvinyl alcohols, dextrins, sugars and vegetable oils). One or moreother additives may also be included in granules (for example anemulsifying agent, wetting agent or dispersing agent).

Dispersible Concentrates (DC) may be prepared by dissolving a compoundof the invention in water or an organic solvent, such as a ketone,alcohol or glycol ether. These solutions may contain a surface activeagent (for example to improve water dilution or prevent crystallizationin a spray tank).

Emulsifiable concentrates (EC) or oil-in-water emulsions (EW) may beprepared by dissolving a compound of the invention in an organic solvent(optionally containing one or more wetting agents, one or moreemulsifying agents or a mixture of said agents). Suitable organicsolvents for use in ECs include aromatic hydrocarbons (such asalkylbenzenes or alkylnaphthalenes, exemplified by SOLVESSO 100,SOLVESSO 150 and SOLVESSO 200; SOLVESSO is a Registered Trade Mark),ketones (such as cyclohexanone or methylcyclohexanone) and alcohols(such as benzyl alcohol, furfuryl alcohol or butanol),N-alkylpyrrolidones (such as N-methylpyrrolidone or N-octylpyrrolidone),dimethyl amides of fatty acids (such as C₈-C₁₀ fatty acid dimethylamide)and chlorinated hydrocarbons. An EC product may spontaneously emulsifyon addition to water, to produce an emulsion with sufficient stabilityto allow spray application through appropriate equipment. Preparation ofan EW involves obtaining a compound of the invention either as a liquid(if it is not a liquid at room temperature, it may be melted at areasonable temperature, typically below 70° C.) or in solution (bydissolving it in an appropriate solvent) and then emulsifiying theresultant liquid or solution into water containing one or more SFAs,under high shear, to produce an emulsion. Suitable solvents for use inEWs include vegetable oils, chlorinated hydrocarbons (such aschlorobenzenes), aromatic solvents (such as alkylbenzenes oralkylnaphthalenes) and other appropriate organic solvents which have alow solubility in water.

Microemulsions (ME) may be prepared by mixing water with a blend of oneor more solvents with one or more SFAs, to produce spontaneously athermodynamically stable isotropic liquid formulation. A compound of theinvention is present initially in either the water or the solvent/SFAblend. Suitable solvents for use in MEs include those hereinbeforedescribed for use in ECs or in EWs. An ME may be either an oil-in-wateror a water-in-oil system (which system is present may be determined byconductivity measurements) and may be suitable for mixing water-solubleand oil-soluble pesticides in the same formulation. An ME is suitablefor dilution into water, either remaining as a microemulsion or forminga conventional oil-in-water emulsion.

Suspension concentrates (SC) may comprise aqueous or non-aqueoussuspensions of finely divided insoluble solid particles of a compound ofthe invention. SCs may be prepared by ball or bead milling a solidcompound of the invention in a suitable medium, optionally with one ormore dispersing agents, to produce a fine particle suspension of thecompound. One or more wetting agents may be included in the compositionand a suspending agent may be included to reduce the rate at which theparticles settle. Alternatively, a compound of the invention may be drymilled and added to water, containing agents hereinbefore described, toproduce the desired end product.

Aerosol formulations comprise a compound of the invention and a suitablepropellant (for example n-butane). A compound of the invention may alsobe dissolved or dispersed in a suitable medium (for example water or awater miscible liquid, such as n-propanol) to provide compositions foruse in non-pressurized, hand-actuated spray pumps.

A compound of the invention may be mixed in the dry state with apyrotechnic mixture to form a composition suitable for generating, in anenclosed space, a smoke containing the compound.

Capsule suspensions (CS) may be prepared in a manner similar to thepreparation of EW formulations but with an additional polymerizationstage such that an aqueous dispersion of oil droplets is obtained, inwhich each oil droplet is encapsulated by a polymeric shell and containsa compound of the invention and, optionally, a carrier or diluenttherefor. The polymeric shell may be produced by either an interfacialpolycondensation reaction or by a coacervation procedure. Thecompositions may provide for controlled release of the compound of theinvention and they may be used for seed treatment. A compound of theinvention may also be formulated in a biodegradable polymeric matrix toprovide a slow, controlled release of the compound.

A composition may include one or more additives to improve thebiological performance of the composition (for example by improvingwetting, retention or distribution on surfaces; resistance to rain ontreated surfaces; or uptake or mobility of a compound of the invention).Such additives include surface active agents, spray additives based onoils, for example certain mineral oils or natural plant oils (such assoybean and canola oil), and blends of these with other bio-enhancingadjuvants (ingredients which may aid or modify the action of a compoundof the invention).

A compound of the invention may also be formulated for use as a seedtreatment, for example as a powder composition, including a powder fordry seed treatment (DS), a water soluble powder (SS) or a waterdispersible powder for slurry treatment (WS), or as a liquidcomposition, including a flowable concentrate (FS), a solution (LS) or acapsule suspension (CS). The preparations of DS, SS, WS, FS and LScompositions are very similar to those of, respectively, DP, SP, WP, SCand DC compositions described above. Compositions for treating seed mayinclude an agent for assisting the adhesion of the composition to theseed (for example a mineral oil or a film-forming barrier).

Wetting agents, dispersing agents and emulsifying agents may be surfaceSFAs of the cationic, anionic, amphoteric or non-ionic type.

Suitable SFAs of the cationic type include quaternary ammonium compounds(for example cetyltrimethyl ammonium bromide), imidazolines and aminesalts.

Suitable anionic SFAs include alkali metals salts of fatty acids, saltsof aliphatic monoesters of sulfuric acid (for example sodium laurylsulfate), salts of sulfonated aromatic compounds (for example sodiumdodecylbenzenesulfonate, calcium dodecylbenzenesulfonate,butylnaphthalene sulfonate and mixtures of sodium di-isopropyl- andtri-isopropyl-naphthalene sulfonates), ether sulfates, alcohol ethersulfates (for example sodium laureth-3-sulfate), ether carboxylates (forexample sodium laureth-3-carboxylate), phosphate esters (products fromthe reaction between one or more fatty alcohols and phosphoric acid(predominately mono-esters) or phosphorus pentoxide (predominatelydi-esters), for example the reaction between lauryl alcohol andtetraphosphoric acid; additionally these products may be ethoxylated),sulfosuccinamates, paraffin or olefin sulfonates, taurates andlignosulfonates.

Suitable SFAs of the amphoteric type include betaines, propionates andglycinates.

Suitable SFAs of the non-ionic type include condensation products ofalkylene oxides, such as ethylene oxide, propylene oxide, butylene oxideor mixtures thereof, with fatty alcohols (such as oleyl alcohol or cetylalcohol) or with alkylphenols (such as octylphenol, nonylphenol oroctylcresol); partial esters derived from long chain fatty acids orhexitol anhydrides; condensation products of said partial esters withethylene oxide; block polymers (comprising ethylene oxide and propyleneoxide); alkanolamides; simple esters (for example fatty acidpolyethylene glycol esters); amine oxides (for example lauryl dimethylamine oxide); and lecithins.

Suitable suspending agents include hydrophilic colloids (such aspolysaccharides, polyvinylpyrrolidone or sodium carboxymethylcellulose)and swelling clays (such as bentonite or attapulgite).

A compound of the invention may be applied by any of the known means ofapplying pesticidal compounds. For example, it may be applied,formulated or unformulated, to the pests or to a locus of the pests(such as a habitat of the pests, or a growing plant liable toinfestation by the pests) or to any part of the plant, including thefoliage, stems, branches or roots, to the seed before it is planted orto other media in which plants are growing or are to be planted (such assoil surrounding the roots, the soil generally, paddy water orhydroponic culture systems), directly or it may be sprayed on, dustedon, applied by dipping, applied as a cream or paste formulation, appliedas a vapor or applied through distribution or incorporation of acomposition (such as a granular composition or a composition packed in awater-soluble bag) in soil or an aqueous environment. A compound of theinvention can be applied topically to protect humans or animals, e.g.,cattle, horses, dogs, cats, sheep, goats, etc., from biting insects orticks.

A compound of the invention may also be injected into plants or sprayedonto vegetation using electrodynamic spraying techniques or other lowvolume methods, or applied by land or aerial irrigation systems.

Compositions for use as aqueous preparations (aqueous solutions ordispersions) are generally supplied in the form of a concentratecontaining a high proportion of the active ingredient, the concentratebeing added to water before use. These concentrates, which may includeDCs, SCs, ECs, EWs, MEs, SGs, SPs, WPs, WGs and CSs, are often requiredto withstand storage for prolonged periods and, after such storage, tobe capable of addition to water to form aqueous preparations whichremain homogeneous for a sufficient time to enable them to be applied byconventional spray equipment. Such aqueous preparations may containvarying amounts of a compound of the invention (for example 0.0001 to10%, by weight) depending upon the purpose for which they are to beused.

Analogs of VU041, VU937, and of Other Kir Channel inhibitors.

Among the compounds that may be used in practicing the present inventionare not only of VU041 and VU937, but also analogs of VU041 and VU937.Among those analogs are the several compounds listed in Appendices A andB of priority application 62/422,382, each of which has chemicalsimilarity to VU041 or VU937. The complete disclosure of priorityapplication 62/422,382, including its Appendices A and B, is herebyincorporated by reference in its entirety.

Among the compounds that may be used in practicing the present inventionare the specific compounds listed in Appendix A or Appendix B ofpriority application 62/422,382. Also among the compounds that may beused in practicing the present invention are compounds whose structureis the same as that of VU041 or VU937, or one of the compounds listed inAppendix A or Appendix B of priority application 62/422,382, or one ofthe Kir channel inhibitors otherwise described in the presentspecification; but with one, two, three, or four of the following typesof substitutions or modifications made to the structure: replacing afluorine atom with a hydrogen, bromine, chlorine, or iodine atom, orwith a hydroxyl group, methoxy group, or ethoxy group; replacing anitrogen atom with a phosphorus atom; replacing an oxygen atom with asulfur atom; replacing a carbonyl group (C═O) with a carbon atom bondedto a hydroxyl group, thiol group, methoxy group, or ethoxy group (CH—OHor CH—SH or CH—OCH₃ or CH—OCH₂CH₃), replacing a single bond with adouble bond; replacing a double bond with a single bond; replacing ahydrogen atom with a fluorine, bromine, chlorine, or iodine atom, orwith a hydroxyl group, methoxy group, or ethoxy group; or replacing ahydrogen atom with a substituted or unsubstituted methyl, ethyl, propyl,or isopropyl group. In each case, hydrogen atoms may be added to ordeleted from the structures where appropriate to satisfy ordinaryvalences and bonding properties.

Definitions

For purposes of interpreting the specification and Claims: (1) An“effective amount” of a particular compound or agent is an amount thatwill induce a particular, defined outcome (e.g., mortality, reducedsalivary gland secretion, etc., as defined in context), within 12 hours,in at least 90% of individual arthropods of a particular species thathave not acquired resistance to the agent. Through natural selection,populations that are exposed to an adverse agent will tend to acquireresistance to that agent over time. The “90%” figure in this definitionis made in reference to populations that have not yet acquiredresistance to the agent. The “90%” figure in the definition carries noimplications about how prevalent resistance to the agent may have becomeat any given time.

(2) An “exogenous” agent is a compound or a composition to which aparticular species (e.g., Aedes aegypti, Amblyomma americanum, etc.) isnot exposed in a state of nature—whether because the agent isartificial, or because the species does not come into contact with theagent in a state of nature (even if the agent is naturally occurring).In other words, by this definition either the “exogenous” agent itselfis not a “product of nature,” or the process of contacting individualsof the particular species with the particular “exogenous” agent is not aprocess that occurs in a state of nature. By contrast, if the particularagent is naturally occurring, and if individuals of the particularspecies come into contact with that agent in a state of nature, then theagent is not considered “exogenous” within the scope of this definition.

INCORPORATIONS BY REFERENCE

The complete disclosures of all references cited in this specificationare hereby incorporated by reference. Also incorporated by reference isthe complete disclosure of U.S. provisional application Ser. Nos.62/421,621 and 62/422,382. In the event of an otherwise irreconcilableconflict, however, the present specification shall control over materialthat is incorporated by reference.

What is claimed:
 1. A method for inducing an outcome in an arthropod;wherein said arthropod is a mosquito, a tick, an aphid, or a fly; saidmethod comprising: administering to the arthropod an effective amount ofan agent comprising one or more compounds selected from the groupconsisting of pinacidil, VU041, VU063, VU625, and VU730; wherein theoutcome comprises one or more outcomes selected from the groupconsisting of: (i) reducing or destroying salivary gland secretions;(ii) reducing or destroying feeding or digestion; (iii) reducing ordestroying the ability to osmoregulate; (iv) reducing or destroying theability to transmit pathogens; and (v) death of the arthropod; andwherein an effective amount of the agent is an amount that will inducethe outcome, within 12 hours, in at least 90% of individual arthropodsof the same species that have not acquired resistance to the agent. 2.The method of claim 1, wherein the agent is in water-soluble form or theagent is in aqueous suspension; and wherein said administering stepcomprises spraying an aqueous solution or aqueous suspension of theagent onto a plant; wherein the plant thereafter absorbs the agentsystemically; and wherein the agent is thereafter delivered to aphids asthe aphids feed upon the plant.
 3. The method of claim 1, wherein thearthropod is a mosquito.
 4. The method of claim 1, wherein the arthropodis a tick.
 5. The method of claim 1, wherein the arthropod is an aphid.6. The method of claim 1, wherein the arthropod is a fly.
 7. The methodof claim 1, wherein the agent is pinacidil.
 8. The method of claim 1,wherein the agent is VU041.
 9. The method of claim 1, wherein the agentis VU063.
 10. The method of claim 1, wherein the agent is VU625.
 11. Themethod of claim 1, wherein the agent is VU730.
 12. A method for inducingan outcome in an arthropod; wherein said arthropod is an insect or anarachnid; and wherein said arthropod is: (i) hematophagous, or (ii)sap-feeding, or (iii) both; said method comprising: administering to thearthropod an effective amount of an exogenous agent; wherein the agentblocks one or more potassium transport pathways in the arthropod'ssalivary glands; wherein the outcome comprises one or more outcomesselected from the group consisting of: (i) reducing or destroyingsalivary gland secretions; (ii) reducing or destroying feeding ordigestion; (iii) reducing or destroying the ability to osmoregulate;(iv) reducing or destroying the ability to transmit pathogens; and (v)death of the arthropod; and wherein an effective amount of the agent isan amount that will induce the outcome, within 12 hours, in at least 90%of individual arthropods of the same species that have not acquiredresistance to the agent.
 13. The method of claim 12, wherein the agentblocks one or more Kir channels in the arthropod's salivary glands. 14.The method of claim 12, wherein the agent blocks one or more potassiumion channels in the arthropod's salivary glands.
 15. The method of claim12, wherein the agent is selected from the group consisting of Kirchannel modulators, K2P channel modulators, K_(ATP) channel modulators,and KCC channel modulators.
 16. The method of claim 9, wherein the agentcomprises one or more compounds selected from the group consisting ofpinacidil, VU041, VU063, VU625, and VU730.
 17. The method of claim 9,wherein the agent is in water-soluble form or the agent is in aqueoussuspension; and wherein said administering step comprises spraying anaqueous solution of the agent or an aqueous suspension of the agent ontoa plant; wherein the plant thereafter absorbs the agent systemically;and wherein the agent is thereafter delivered to sap-feeding insects asthe insects feed upon the plant.
 18. The method of claim 9, wherein thearthropod is a mosquito, a tick, an aphid, or a fly.